1 Department of Surgery, Beth Israel Deaconess Medical Center, Boston 02215; Departments of 2 Surgery and 3 Medicine, Harvard Medical School, Boston 02115; 4 Medical Services, Massachusetts General Hospital, Charlestown 02129; and 5 Division of Health Sciences and Technology, Harvard Medical School-Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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ABSTRACT |
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Using an in vivo rat model of unilateral renal ischemia, we previously showed that the expression and distribution of fibronectin (FN), a major glycoprotein of plasma and the extracellular matrix, dramatically changes in response to ischemia-reperfusion. In the distal nephron in particular, FN accumulates in tubular lumens, where it may contribute to obstruction. In the present study, we examine whether the tubular FN is the plasma or cellular form, each of which is produced by alternative splicing of a single gene transcript. We demonstrate that FN in tubular lumens does not contain the extra type III A (EIIIA) and/or the extra type III B (EIIIB) region, both of which are unique to cellular FN. It does, however, contain the V95 region, which in the rat is a component of FNs in both plasma and the extracellular matrix. Expression of FN containing EIIIA increases dramatically in the renal interstitium after ischemic injury and continues to be produced at high levels 6 wk later. V95-containing FN also increases in the interstitial space, albeit more slowly and at lower levels than FN containing EIIIA; it also persists 6 wk later. FN containing the EIIIB region is not expressed in the injured kidney. The presence of V95 but not the EIIIA or EIIIB regions of FN in tubular lumens identifies the origin of FN in this location as the plasma; tubular FN is ultimately voided in the urine. The data indicate that both plasma and cellular FNs containing the V95 and/or EIIIA regions may contribute to the pathogenesis of acute renal failure and to the repair of the injured kidney.
alternative splicing; extracellular matrix; renal injury; regeneration; acute renal failure
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INTRODUCTION |
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FIBRONECTINS
(FNs) ARE A FAMILY of large (~440 kDa) dimeric
glycoproteins implicated in a variety of biological processes, including tissue remodeling during wound healing and embryonic development (for review, see Refs. 29, 40). FNs influence these
processes by affecting cell adhesion and extracellular matrix assembly.
They exist in two forms, plasma and cellular. Plasma FN is made by
hepatocytes and circulates in the blood; cellular FN is produced by
epithelial and connective tissue cells and is deposited into the
extracellular matrix. Both forms of FN are encoded by a single gene,
whose transcript can be alternatively spliced at three intron-exon
boundaries, yielding FN protein subunits containing, in the rat, the
extra type III A (EIIIA) repeat, the extra type III B (EIIIB) repeat,
and/or the variable (V) region (Fig. 1;
for review, see 19, 51-52). Equivalent regions in humans are designated EDA, EDB, and IIICS. During splicing of FN mRNA, these
exons are either included or excluded depending on the tissue and cell
type. In plasma FN mRNA, exons encoding the EIIIA and EIIIB segments
are excluded, whereas the exon encoding the V region is either
partially or completely included depending on the species. In cellular
FN mRNA, EIIIA and/or EIIIB exons are wholly retained and the V region
appears to be at least partially included (43, 45, 56). Up
to 12 different combinations of the three alternatively spliced
segments are possible, resulting in a heterogeneous mixture of FN
isoforms (32, 53).
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The function of the various isoforms is incompletely understood. FNs retaining the EIIIA and/or EIIIB regions are widely expressed in embryonic tissues and cultured cell lines compared with normal adult tissues (12, 20-21, 43-44, 46-47, 53, 64). Upregulation of EIIIA- and/or EIIIB-containing FN has also been noted in the adult at sites of tissue injury, such as in the skin (14, 22), eye (41, 67), and vasculature (17, 25, 28, 60). Isoforms containing a subsegment of the V region known as V25 are also preferentially expressed in embryos and injured peripheral nerves (37, 68).
In the kidney, FN isoforms are differentially expressed during development. In human fetal kidney, FN containing the EDA segment is widely found in tubular and glomerular basement membranes, the interstitium, and glomerular mesangium (34). The isoform containing the EDB region follows a similar but more restricted distribution, localizing to the mesangium, the interstitium, and basement membranes of medullary tubules and collecting ducts, albeit at lower levels (34). In the adult human nephron, FN containing EDA is found only in the mesangium; neither EDA- nor EDB-containing FNs are detected in tubular basement membranes or the interstitial compartment (34).
We previously demonstrated in an in vivo model of unilateral renal ischemia that FN strongly increases in expression and changes its distribution in response to injury (76). In particular, high concentrations of FN were observed in tubular lumens, suggesting a possible role in tubular obstruction. This study was initiated to understand whether tubular FN originated from plasma or from epithelial and/or connective tissue cells after ischemic injury. We demonstrate that luminal FN originates from plasma and is voided in the urine. We also observe that in response to ischemia-reperfusion, FN containing the EIIIA and/or V segments dramatically increases and V-FN, in particular, changes its distribution. These observations have important implications for understanding the mechanism(s) involved in the pathophysiology of acute renal failure (26, 62).
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MATERIALS AND METHODS |
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Surgical procedure. A unilateral rat model of renal ischemia in which the renal artery and vein are clamped for 40 min whereas the contralateral kidney is left intact was used in this study.
Male Sprague-Dawley rats (190-275; Charles River Breeding Laboratories, Wilmington, MA) were housed for 6 days before surgery in the animal facility under alternating cycles of 12 h light, 12 h dark. Water and rat chow were administered ad libitum. In preparation for surgery involving unilateral renal ischemia without removal of the contralateral kidney, animals were weighed and 5-10 ml of sterile saline at 37°C were injected intraperitoneally. Animals were then anesthesized with pentobarbital sodium (6.5 mg/100 g; Abbott Laboratories, North Chicago, IL), and shaved over the left flank. Each was positioned on a warm operating platform (Homeothermic Control Unit; Harvard Apparatus, Holliston, MA) maintained at 37°C, and an incision was made over the left flank region. The renal artery, vein, and ureter were dissected free of surrounding tissue; only the renal artery and vein were clamped with a microaneurysm clamp (Roboz Surgical Instruments, Washington, DC). The incision was temporarily closed, and the animal was placed in an enclosed chamber at 37°C (Small Animal Intensive Care Unit, Harvard Apparatus). After 40 min, the clamp was removed, allowing the kidney to be reperfused with blood. The incision was then permanently sutured, and, after recovery, animals were placed in metabolic cages (Nalgene Labware, Rochester, NY). At the stated times after reperfusion, both kidneys (left postischemic and right contralateral) were either fixed in situ for immunocytochemistry or excised and frozen in liquid nitrogen for biochemistry (see Tissue harvesting). Some animals underwent sham surgery without ischemia. For sham surgery, in lieu of clamping the renal artery and vein, the left kidney was surgically exposed and gently manipulated. In the unilateral model of renal ischemia, there are only mild systemic changes in blood urea nitrogen and creatinine because of the presence of the intact contralateral kidney (36, 72).Urine collection.
Urine was collected at 24-h intervals in the presence of Complete
protease inhibitors (Boehringer-Mannheim, Indianapolis, IN) from
animals housed in metabolic cages (100 µl of 50× protease inhibitor/collection tube). The volume collected was measured, and the
urine was filtered through two layers of cheesecloth before being
centrifuged at 2,000 rpm at 0°C for 30 min. Urine was then filtered
through a 0.45-µm Millex-HA filter (Millipore, Bedford, MA),
aliquoted, and frozen in liquid nitrogen. Samples were stored at
80°C.
Tissue harvesting. Animals were anesthetized before tissue harvesting. For immunocytochemistry, rats were perfused through the left ventricle with warm Hanks' balanced salt solution and then with 2% paraformaldehyde-75 mM L-lysine-10 mM sodium periodate (PLP; 38). Kidneys were removed and stored intact in fixative overnight at 4°C. They were then rinsed in PBS and stored in PBS containing 0.02% sodium azide at 4°C.
For biochemical analysis, rats were perfused through the left ventricle with sterile PBS (Dulbecco's formulation) at 0°C containing 0.16× complete protease inhibitors (Boehringer-Mannheim). Kidneys were removed, placed on ice, and dissected free of adherent connective tissue. Slices were cut, extending from the cortex to the hilus, frozen in liquid nitrogen, and stored atAntibodies. A variety of monoclonal and polyclonal antibodies against FN were used in this study. Rabbit anti-rat FN polyclonal antibody (GIBCO-BRL, Grand Island, NY) was generated against rat plasma FN. This antibody recognizes regions of the FN molecule that are common to both the plasma and cellular forms. It was used to detect all forms of FN in tissue sections (1:750) and immunoblots (1:7,500). It is therefore referred to as anti-total FN antibody. Specificity of the anti-total FN antibody was determined by competition experiments between the antibody and purified antigen (76). Briefly, the anti-total FN antibody was incubated with purified rat plasma fibronectin, and the mixture then applied to tissue sections. After rinsing, sections were processed as described below for immunocytochemistry. Under these conditions, immunostaining was obliterated (76).
Culture supernatant containing mouse monoclonal antibody against the EDA region of human cellular FN was obtained from I. Virtanen (Univ. of Helsinki, Helsinki, Finland; 64). Alternatively, the same purified antibody was purchased from Chemicon International (Temecula, CA). This antibody specifically recognizes the extra domain of human cellular FN (EDA sequence) but also cross-reacts with the rat EIIIA region (Chemicon International; see also Figs. 5-7). Culture supernatant was used undiluted for immunofluorescence whereas purified antibody was used at a 1:1,000 dilution to immunoblot kidney tissue extracts and urine. Rabbit polyclonal antibody R2640 to the rat EIIIB segment was obtained from R. O. Hynes (46, 47) and used at a 1:100 dilution. This antibody recognizes the EIIIB segment in the context of a bacterial fusion protein because the immunogen was an EIIIB-glutathione S-transferase fusion protein. In order for this antibody to recognize EIIIB in mammalian cells, N-linked carbohydrate was removed from target FN by pretreating tissue sections overnight with N-glycanase (46, 47; see cryosectioning and immunofluorescence). Rabbit polyclonal antibody 73N recognizing the V95 region of FN was also obtained from R. O. Hynes (46, 47) and was used at a dilution of 1:100 for immunocytochemistry. This region is one of three variants of the V segment found in rat (V120, V95, and V0). Both the rabbit polyclonal antibody R2640 to the rat EIIIB segment and rabbit polyclonal antibody 73N to the V95 region of FN are suitable for immunocytochemistry (7, 42, 46, 47) and not biochemistry. These antibodies have been used for studies in the kidney (7, 42, 46).Cryosectioning and immunofluorescence.
For frozen sections, fixed intact kidneys were sliced and equilibrated
overnight at 4°C in 0.6 M sucrose/PBS. Kidney slices were then
embedded in OCT (optimal cutting temperature) compound (Miles
Laboratories, Naperville IL) and frozen in liquid nitrogen. Sections (5 µm) were cut on either a Reichert Frigocut cryostat (Leica,
Deerfield, IL) or a Leica CM1850 cryostat. Sections were placed on
SuperFrost Plus glass slides (Fisher Scientific, Boston, MA) and stored
at 20°C until use.
Western Blotting
Immunoblotting was repeated at least three times with samples from a minimum of two animals for each time point and condition.Tissue samples. Frozen kidney tissue was quickly weighed and transferred to a Dounce homogenizer on ice. Nine parts 2× sample buffer (400 mM Tris, pH 8.8, 10 mM EDTA, 2% SDS) at 4°C containing protease inhibitors (10 µg/ml aprotinin, 1 µg/ml antipain, 1 µg/ml pepstatin, 17.5 µg/ml benzamidine) but lacking sucrose, bromophenol blue and dithiothreitol (DTT) were then added. Tissue was extensively homogenized and boiled for a total of 8 min at 95°C to completely solubilize cell proteins. Samples were then cooled to room temperature, DTT was added to 40 mM, and samples were further homogenized and boiled (4 min). After cooling to room temperature, samples were sheared in succession with 22-, 23-, and 26-gauge needles, transferred to microfuge tubes, and spun twice at 16,000 g for 5 min at room temperature. Any remaining pellet was reextracted (2× sample buffer containing DTT, SDS, and protease inhibitors), sheared, and centrifuged as before. Supernatants were pooled, and protein concentration was then measured using the Standard Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with BSA as standard.
Urine samples. Urine samples were thawed on ice for 15-20 min and centrifuged for 5 min at 4°C. Typically, no pellet was visible. Volumes of urine containing equivalent amounts of protein were mixed with an equal volume of 2× sample buffer containing 24% sucrose, 0.2% bromophenol blue, 40 mM DTT and protease inhibitors. Samples were boiled at 95°C for 4 min and quickly cooled on ice.
Tissue and urine samples were further diluted with 1× sample buffer (200 mM Tris, pH 8.8, 5 mM EDTA, 12% sucrose, 0.1% bromophenol blue, 20 mM DTT, 2% SDS) without protease inhibitors to yield aliquots of protein suitable for gel loading. They were then boiled again, cooled, alkylated with 0.5 M iodoacetamide in the dark at 37°C for 20 min, and either frozen in liquid nitrogen and stored at ![]() |
RESULTS |
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We and others have previously demonstrated in in vivo rat models of renal ischemia that cell extracellular matrix interactions dynamically change in response to injury (70, 76). In particular, we demonstrated that FN, a major glycoprotein of the plasma and renal interstitium, significantly altered its distribution and expression. Most surprisingly, it was intensely immunostained in tubular lumens. In this location, FN might contribute to tubular obstruction, a major outcome of renal artery occlusion and reperfusion. Because our earlier work did not distinguish among the various forms of FN, we were unable to determine whether lumenal FN originated from leakage of plasma proteins or from the synthesis and deposition of the cellular form by tubular epithelial or connective tissue cells. Resolution of this issue is important for our understanding of how FN is involved in the pathogenesis of ischemic renal injury. To investigate the source of luminal FN, polyclonal and monoclonal antibodies against specific regions of this molecule that are found in either plasma and/or cellular FNs (EIIIA, EIIIB, and V95) were used to determine the distribution of plasma and cellular FNs in response to ischemia reperfusion.
Distribution and Expression of FN in the Outer and Inner Stripes
Total FN.
Using a polyclonal antibody that recognizes both plasma and cellular
FNs, referred to as anti-total FN antibody, FN was immunolocalized in
kidney sections of rats killed at various times after reperfusion after
a 40-min unilateral clamp of the renal artery and vein. In kidneys made
ischemic but not reperfused (0 h postischemia, Fig.
2, A and B),
anti-total FN antibody outlines tubules in the outer stripe (Fig.
2A) and inner stripe (Fig. 2B); it also stains the interstitial space (Figs. 2, A and B). One to
three hours after reperfusion, localization is maintained (outer
stripe, Fig. 2C; inner stripe, Fig. 2D). However,
anti total-FN antibody also stains the luminal space of some tubules of
the thick ascending limb in the inner stripe (asterisks, Fig.
2D).
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EIIIA-FN.
At 0 h postischemia, FN containing the EIIIA segment is
not detected in the outer (Fig.
5A) or inner stripes (Fig.
5B) of the outer medulla. Rarely, some patchy, faint
immunostaining associated with tubular basement membranes is observed
(arrow, Fig. 5A); red blood cells also autofluoresce at this
time (asterisks; Fig. 5, A and B). Production of
EIIIA-FN significantly increases 1-3 h after reperfusion,
associating primarily with basement membranes of S3 proximal tubules in
the outer stripe (Fig. 5C) and thick limbs of the inner
stripe (Fig. 5D). Interstitial localization in the inner
stripe is also strong. FN containing the EIIIA segment is not detected
in tubular lumens of the inner stripe (Fig. 5D), in contrast
to the observation with anti-total FN antibody (Fig. 2D).
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EIIIB-FN. FN containing the EIIIB region was examined next. EIIIB, like EIIIA, is unique to cellular FN. EIIIB-FN was not detected in kidney sections at any of the time points examined after ischemic injury. It is also not detected in normal adult rat (42, 44), mouse (46), or human (34) kidneys. As a positive control for immunostaining, EIIIB-FN was detected in Descemet's membrane of the adult rat cornea (data not shown; 46).
V95-FN.
Localization of the V95 region of FN, which in the rat is found in a
fraction of both plasma and cellular FNs (54, 55), was
also examined. At 0 h postischemia, little if any FN
containing the V95 region is detected in the outer (arrows, Fig.
8A) and inner stripes (Fig.
8B). One to three hours later, localization in the outer
stripe (Fig. 8C) does not change; however, in the inner
stripe (Fig. 8D), FN containing V95 begins to localize to some lumenal compartments (asterisks, Fig. 8D).
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Distribution and Expression of FN Isoforms in the Cortex
FN isoforms were also immunolocalized in the cortex after ischemic injury to determine whether either expression or distribution in this region was affected, as in the outer and inner stripes. We also examined whether the immunostaining pattern provided evidence of glomerular damage, which could explain the source of luminal FN in both this and our previous study (76). Glomerular injury would disrupt the glomerular filtration barrier, allowing access of plasma proteins (greater than 70 kDa) such as FN into Bowman's space.Total FN.
At 0 (Fig. 9A), 1, 3 (Fig.
9B), 24, 48 (Fig. 9C), and 120 h (Fig.
9D) postischemia, anti-total FN antibody highlights
Bowman's capsule and the glomerular capillary tuft. Total FN is not
observed in the urinary space of renal corpuscles at any time
(asterisk, Figs. 9, A-D); however, from 3 to 48 h after
reperfusion, it is more frequently detected in tubular lumens of the
distal nephron adjacent to glomeruli (double asterisk, Figs. 9,
B and C, respectively). The latter observation of
FN in tubular lumens of nephron segments distal to glomeruli is
consistent with results from the outer (see Fig. 2E) and
inner stripes (see Fig. 2, D and F).
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EIIIA-FN.
Immunostaining of both glomeruli and the interstitium of the cortex was
examined with antibody recognizing the EIIIA region of FN. In
glomerular regions at 0 h postischemia, anti-EIIIA
antibody faintly demarcates the connective tissue of the capillary tuft (Fig. 11A; 34). This
staining pattern does not change 1-3 h after reperfusion (data not
shown). However, 24-48 h after injury (Fig. 11B),
components of the renal corpuscle, including Bowman's capsule, stain
more intensely, persisting at 120 h postischemia (Fig.
11C). FN containing EIIIA does not localize to Bowman's
space at any time after reperfusion (asterisk, Figs. 11,
A-C) and is absent from tubular lumens of the
distal nephron.
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EIIIB-FN. The FN isoform containing the EIIIB region was not detected in the cortex either before (42) or after ischemic injury (data not shown).
V95-FN. Distribution and expression of FN containing the V95 region was examined in the glomerular and interstitial regions of the cortex. In the glomerular region at 0 (Fig. 11D), 3 (data not shown), 48 (Fig. 11E), and 120 h (Fig. 11F) postischemia, V95-FN weakly localizes to the connective tissue surrounding the capillary tuft and steadily increases in Bowman's capsule. FN containing V95 is absent from the urinary space at all times after reperfusion (asterisk, Fig. 11, D-F) but is sometimes seen in the lumen of the distal nephron between 3 and 48 h after reperfusion (data not shown).
V95-FN also localizes to the cortical interstitium. From 0 (Fig. 11D) to 3 h (data not shown) postischemia, immunostaining with the anti-V95-FN antibody is rarely detected, consistent with our observation in the outer and inner stripes at this time (see Fig. 8, A-D). However, at 24-48 h postischemia, V95-FN increases in the interstitial compartment surrounding tubular basement membranes (Fig. 11E). A gradient of staining, ranging from greater intensity in cortical areas adjacent to the outer medulla to lesser intensity in cortical regions distant from it, is not readily detected, in contrast to the immunstaining pattern observed for EIIIA-FN (see Fig. 11B). At 120 h postischemia, V95-FN is not present to the same extent as EIIIA (see Fig. 11C) or total FN (see Fig. 9D). Antibody recognizing V95 strongly outlines only some tubules in this location (Fig. 11F). In contralateral kidneys at all time points, FN containing V95 weakly localizes to the connective tissue of the capillary tuft and to Bowman's capsule (data not shown), as in the postischemic injured kidney at 0 h (see Fig. 11D). None is detected in the luminal space at any time (data not shown). Faint, patchy immunostaining is infrequently seen in the interstitial compartment (data not shown). A similar pattern is observed in cortical regions of mock kidneys at all time points, except that interstitial staining is barely detectable (data not shown). Thus in the cortex after ischemic injury, no FN isoforms are detected in the urinary space of the glomerulus. In addition, the FN isoform containing EIIIA and, to a lesser extent, V95-FN develops a gradient of expression, with more localizing in cortical regions near the outer medulla and less in regions distant from it. Both isoforms localize to the interstitium surrounding tubular basement membranes. FN containing EIIIB is not detected.Plasma But Not Cellular FN is Voided in the Urine
Immunostaining with anti-total FN and anti-V95 antibodies indicated that FN localized to tubular lumens shortly after reperfusion (1-3 h) peaked when injury predominated (24-48 h) and later disappeared as regeneration continued (120 h reperfusion; see Figs. 2 and 8, respectively; 76). Loss of FN from tubular lumens could be due to excretion. To confirm this, urine collected at 24-h intervals from animals that had undergone unilateral renal ischemia was analyzed for the presence of plasma and/or cellular FN. Urine from mock-operated animals served as control. Urine containing equal amounts of protein were electrophoresed, and SDS gels were immunoblotted with either anti-total or anti-cellular FN antibody recognizing human EDA.Immunoblotting with the anti-total FN antibody demonstrates that FN is
voided in the urine in response to ischemia-reperfusion injury.
Two patterns emerge. In some animals (n = 3), total FN is voided at increasing levels (Fig.
12A), beginning as early as 24 h after reperfusion, peaking 72-96 h after reperfusion and in some instances declining thereafter. In other animals
(n = 4), a biphasic excretion pattern is seen (Fig.
12A). Total FN is voided at high levels 24 h after
reperfusion, declines 48-72 h after reperfusion, and again
increases from 72-120 h, reaching levels equivalent to that at the
24-h time point. In urine of mock operated or normal animals, total FN
is excreted at very low levels (Fig. 12A).
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In contrast to the data with the anti-total FN antibody, immunoblotting with anti-cellular EDA antibody confirms that cellular FN synthesized by renal epithelia or connective tissue cells is not detected in the urine at any time after ischemia-reperfusion (Fig. 12B). It is also not detected in urine collected from mock-operated animals (Fig. 12B). Thus cellular FN is not excreted, suggesting that all the FN that is excreted is from the plasma and that it is plasma not cellular FN that is found in tubular lumens.
Distribution and Expression of FN Isoforms 6 Wk After Ischemic Injury
The observation that total FN-, EIIIA-, and V95-containing FN isoforms persist at high levels 120 h after reperfusion when repair from ischemic injury is almost complete and redifferentiation of the damaged tubular epithelium predominates (1, 72, 76) raised the question of whether the distribution and expression of FN isoforms containing EIIIA and/or V95 persists at later times.At 6 wk postischemia, FN continues to localize at high levels
in previously injured regions of the outer medulla, identified by the
heterogeneous shapes and sizes of the regenerated tubules. Antibody
against total FN strongly stains the interstitial compartment, sometimes concentrating at higher levels adjacent to basement membranes
of renal tubules (Fig. 13A).
In contrast, antibodies to EIIIA and V95-FNs mainly identify tubular
basement membranes of varying thicknesses, whereas other basement
membranes are not outlined (Fig. 13, B-C,
respectively). The interstitial compartment contains some EIIIA and
V95-FN, however, at significantly lower levels than that observed for
total FN.
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In the cortex, FN containing the EIIIA or V95 regions is not detected in association with basement membranes or the interstitium (data not shown); sclerosis of glomeruli also is not evident (data not shown).
FN containing EIIIB is not detected in either the outer medulla or cortex of the 6-wk postischemic kidney (data not shown).
Thus EIIIA and V95-FNs are found in the 6-wk postischemic kidney, localizing to the basement membrane region of previously injured tubules. They are also found to a lesser extent in the surrounding interstitium.
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DISCUSSION |
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We demonstrate using an in vivo model of unilateral renal
ischemia that production of FN splice variants dramatically
changes as a result of renal injury and repair (Table
1). In ischemic kidneys that are
not reperfused (0 h), the alternatively spliced forms of FN,
which contain the EIIIA, EIIIB, and/or V95 regions, are absent from the
renal interstitium. However, at early times after reperfusion (1-3
h), FN containing EIIIA increases in this location in the outer medulla
whereas FN containing V95 does not. The isoform containing V95, but not
the EIIIA or EIIIB region, is found in a few lumens of the distal
nephron. At 24-48 h postischemia, as further injury and
regeneration coordinately occur, localization of both EIIIA- and
V95-containing FN isoforms increases in the interstitial matrix in both
the outer medulla and cortex, with the increase in EIIIA-FN being more
dramatic. The V95-containing FN isoform, but not that containing EIIIA,
also frequently fills the luminal space. As regeneration continues at
120 h after reperfusion, EIIIA- and V95-containing FN strongly
localize to the interstitial compartment, and V95-FN is no longer
detected in tubular lumens. Luminal FN is ultimately excreted in the
urine. Six weeks after ischemic injury, FN containing EIIIA
and/or V95 persists around some renal tubules.
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Our data using polyclonal and monoclonal antibodies specific for unique regions of the various isoforms of FN indicate that luminal FN originates from plasma. FN in tubular spaces was detected by immunohistochemistry with antibodies recognizing both total and V95-FNs but not with antibodies against the EIIIA- or EIIIB-containing cellular isoforms. In addition, FN excreted in the urine was not detected by antibody specific for cellular FN.
It is unclear how plasma FN enters the luminal space. There is no evidence that it is made by other cell types in addition to hepatocytes (61). Furthermore, our data suggest that luminal plasma FN does not originate from damage to the glomerular filtration barrier, which normally is impermeant to plasma proteins. Total or V95-FNs, with molecular weights of ~440 kDa, were not detected in Bowman's space at any time after ischemia. We believe it likely, therefore, that plasma proteins, including FN, extravasate from interstitial blood vessels into the interstitial space from where it penetrates damaged tubules and enters the lumen. Permeability changes in interstitial blood vessels would allow the escape of plasma FN into the interstitium. In combination with tubulorrhexis and/or disruption to the epithelial permeability barrier (59, 69), plasma FN would then gain entry into tubular lumens. Once in the lumen of the injured tubule, plasma FN concentrates in more distal nephron segments.
Under these circumstances, luminal plasma FN might play a very significant role in the pathophysiology of acute renal failure. Our data indicate that within the first 48 h of ischemic injury, FN is detected by immunohistochemistry in the luminal space of the injured kidney while simultaneously being excreted in the urine. Although we have no evidence plasma FN is occluding the lumen, the high concentration of plasma FN in this location may suggest that its movement is retarded. This may occur through the formation of obstructive complexes with other molecules such as the Heymann antigen gp330 (39) on microvilli shed in response to injury (16, 65) as well as other cellular debris, contributing to the formation of casts. We do not believe plasma FN adheres to sloughed dead or viable cells in the luminal space, because FN fibrils, which assemble on cell-FN interactions (27, 71, 73-74), are not detected. Ultimately, all plasma FN is excreted, indicating that any obstruction is not permanent (see Figs. 2H and 8H).
We also demonstrate that after ischemic injury the FN isoforms containing the EIIIA and V95 regions but not EIIIB localize to the renal interstitium surrounding tubular basement membranes. Before injury, FN containing EIIIA or V95 is not detected in this location. Within 3 h of reperfusion, however, an increase in EIIIA but not V95-FN is observed in the interstitial matrix of the outer medulla and continues to increase at 24-48 h after injury. Although V95-FN increases somewhat at this time, a parallel rise is not detected. Both persist at high levels in the renal interstitium 120 h after reperfusion.
The upregulation of FN isoforms suggests that ischemic injury
may stimulate a signal regulating the production of the alternatively spliced forms. Transforming growth factor- (TGF-
), a
multifunctional cytokine that operates in either a paracrine or
autocrine manner, stimulates fibronectin synthesis (30)
and regulates production of the EIIIA-FN isoform by acting on FN
pre-mRNA (13, 35). It is derived from a number of cell
types including platelets (5), wound macrophages
(49), and fibroblasts/myofibroblasts (75), as
well as injured proximal tubular epithelial cells (10). It
has a known role in the pathogenesis of renal disease (11, 58) and is believed to promote tissue regeneration after renal injury (8, 10). It is unclear which cells at the various time points after reperfusion produce TGF-
in the in vivo model of
unilateral renal ischemia used in this study. Whether TGF-
acts in a paracrine or autocrine fashion on injured epithelial or
connective tissue cells (e.g., infiltrating macrophages,
fibroblasts/myofibroblasts) to promote the synthesis, secretion, and
deposition into the extracellular matrix of FN containing the EIIIA
region also needs to be determined. TGF-
when administered in vitro
increases expression of EIIIA-FN mRNA by proximal tubule cells
(35, 66); its immunoneutralization in vivo in a bilateral
model of renal injury also attenuates the rise in FN containing EIIIA
(9). The production of TGF-
and other paracrine growth
factors may explain the unusual gradient of staining for EIIIA-FN in
the cortex, which is intense in regions adjacent to the outer medulla
(see Fig. 11B). Paracrine growth factors may also be
responsible for the observation that EIIIA-FN is not detected in the
cortex until 24 h after reperfusion (Fig. 11B) even
though in the outer and inner stripes it is present at high levels soon
after reperfusion (1-3 h postischemia; see Fig. 5,
C-D).
Growth factors produced by the injured kidney may also explain the
appearance of the alternatively spliced forms in the contralateral kidney detected by immunostaining. The FN isoform containing EIIIA was
detected within hours in the contralateral kidney, whereas the FN
isoform containing V95 was detected later. Both persisted at the latest
time point we examined, although levels did not increase from that seen
initially and overall were less than that seen in the ischemic
kidney. Perhaps growth factors, such as TGF-1, released by the
injured ischemic kidney enter the systemic circulation; in this
location, it may act on the contralateral kidney to stimulate the
production and accumulation of FN containing EIIIA and/or V95.
Alternatively, the experimental procedure of unilaterally clamping the
renal artery and vein may have resulted in some generalized reduction in blood pressure, resulting in reduced perfusion of the
contralateral kidney.
In the postischemic kidney, the role of FN containing EIIIA is
unknown; however, our data indicate that it may be necessary for
regeneration. EIIIA-FN is widely expressed during organogenesis and
tissue remodelling in the embryo (20-21, 43-44, 47,
64) and during wound healing of various tissue types
(2-3, 7, 14, 17, 20, 25, 28, 41-42, 60, 67). In
each of these cases, FN containing EIIIA may be required for cell
migration, proliferation, and differentiation. The association of
EIIIA-FN with tubular basement membranes may promote migration of
remaining epithelial cells after damaged cells have exfoliated, as well as the spreading of adherent epithelia to fill in denuded areas of
basement membrane. Similarly, it may stimulate epithelial
proliferation, which occurs during this time and is necessary for
repopulation of the damaged tubule (72). These cellular
responses are key elements of epithelial redifferentiation after renal
ischemic injury (62, 76). It would be interesting
to examine at the ultrastructural level whether EIIIA-FN is indeed an
integral component of regenerating basement membranes and whether in
this location it mediates these cell-extracellular matrix interactions,
perhaps by inducing the expression of novel extracellular matrix
receptors, such as the 5
1 integrin, the major fibronectin
receptor, and/or contributing to the altered distribution of other
integrins (76).
FN containing EIIIA may also play a role in the fibrotic response after
renal ischemic injury. In this regard, FN is believed to be the
first extracellular matrix molecule deposited in renal interstitial
fibrosis (18). In the liver, FN containing EIIIA activates
the conversion of normal hepatic lipocytes into myofibroblast-like cells, a critical event in the initiation of fibrogenesis in the liver
(31). In the formation of granulation tissue in wounded skin, EDA FN is necessary for induction of the myofibroblastic phenotype by TGF-1 (57). Whether deposition of FN
containing EIIIA in the postischemic kidney is necessary for
the conversion of stromal fibroblasts and/or perivascular cells into
myofibroblast-like cells (18) is unknown. However, TGF-
mediates this activation (15) and also stimulates
production of FN containing EIIIA (6). We see that in the
injured kidney FN containing EIIIA is expressed soon after reperfusion,
continues to increase in an expanded interstitium, and persists weeks
later in association with thickened basement membranes. Perhaps
deposition of EIIIA-FN is the key extracellular matrix component that
initiates renal fibrosis after ischemic injury.
More is known about the role of FN containing the V95 region, which in the rat is a component of both plasma and cellular FNs. FN exists as a dimeric glycoprotein, and the V95 region must be present in at least one subunit for its secretion to take place (for review, see Refs. 51-52). Our immunostaining data clearly show that although the EIIIA isoform is present in high amounts in the renal interstitium after ischemic injury, levels of V95 are not necessarily comparable. Similar results have been obtained by immunohistochemistry in skeletal and heart muscle, lung parenchyma, small intestine, pancreas, and peripheral nerve (46). This apparent discrepancy could arise from the presence of the V95 region in only one subunit of the FN dimer and/or from differences in antibody sensitivity or epitope masking.
These same reasons could explain why the FN isoform containing the V95 region is not detected in tubular lumens at the same frequency as total FN, which recognizes both the plasma and cellular forms. The V95 region in the rat is one of three variants produced by alternative splicing of the V region exon (for review, see Refs. 19, 51-52). These variants include V120, which contains all V120 region amino acids, V95, which lacks the first 25 amino acids, and V0, which lacks the entire V region. In rat plasma FN, ~50% of the FN subunits are V0 and 50% are a combination of V120 and V95 (54). Considering that less than 50% of the FN subunits circulating in plasma are V95, it is not surprising that the frequency of the V95 isoform in tubular lumens is not equivalent to that observed with the total FN antibody.
At 6 wk postischemic injury, FN isoforms containing EIIIA and V95 strongly localize to thickened basement membranes of some tubules that initially were damaged and appear incompletely regenerated; localization to the surrounding interstitium is weak. Staining with anti-total FN antibody indicates that total FN is found in tubular basement membranes and is strongly expressed in the interstitial compartment. The data suggest incomplete recovery of some regions of the postischemic kidney. In addition, plasma FN, possibly escaping from interstitial blood vessels, appears to be trapped in the connective tissue compartment, having been deposited into the extracellular matrix. It is possible that plasma FN is prevented from entering the luminal space either because the thickened basement membranes, containing EIIIA and V95 FNs, inhibit its passage and/or the epithelial permeability barrier is reestablished.
Our observation of incomplete recovery 6 wk after unilateral ischemic injury may be clinically relevant. In the patient population, the postischemic kidney has a tremendous capacity to completely regenerate, restoring normal renal function. However, it is possible that some nephrons do not completely recover. We believe that our observation in the unilateral model showing incomplete repair of some nephron segments could indicate heterogeneity of nephron function postischemia (4, 23). Long-term effects could result in chronic renal failure, glomerulosclerosis, and tubulo-interstitial damage (63). Similar processes may ultimately occur in patients suffering from ischemic injury.
In conclusion, we show that in response to ischemic injury, the FN isoforms containing the EIIIA and V95 regions that are not normally expressed in the renal interstitium are significantly upregulated, suggesting a possible dual role in regeneration of the damaged kidney and in fibrosis. Tubular FN originates from plasma and may contribute to obstruction. Determining the role of extracellular matrix molecules in renal ischemic injury and repair and how they interact with damaged and regenerating cells may facilitate our understanding of the pathogenesis of acute renal failure. Knowledge of these processes may contribute to the identification of novel therapeutics aimed at limiting injury or speeding recovery.
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ACKNOWLEDGEMENTS |
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We thank I. Virtanen and R.O. Hynes for antibodies, the Morphology Core of the Harvard Digestive Disease Center (DK-34854) for use of the Leica cryostat, and Dr. Elizabeth D. Hay for use of the Zeiss Axiophot microscope.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-46768 (to K.S. Matlin) and R37-DK-39773 (to J. V. Bonventre).
Address for reprint requests and other correspondence: A. Zuk, Dept. of Surgery, Beth Israel Deaconess Medical Center, Bldg. DA-880, 330 Brookline Ave., Boston, MA 02215 (E-mail: azuk{at}caregroup.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 February 2000; accepted in final form 9 February 2001.
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