By
From the * Center for Inflammatory Diseases, Monash University Department of Medicine, Monash
Medical Centre, 3168, Victoria, Australia; Center for Transgene Technology and Gene Therapy,
Vlaams Interuniversitair Instituut voor Biotechnologie, B-3000 Leuven, Belgium; and § Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland, Ohio 44195
The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.
The plasminogen/plasmin system has important functions in the dissolution of fibrin and in the breakdown
of other extracellular matrix proteins (1). It has pivotal roles
in clot lysis and wound healing (2, 3), but also has the potential to influence the outcome of inflammatory processes,
particularly where fibrin and extracellular matrix deposition
play an important role (4).
Glomerulonephritis (GN) results from immunologically
mediated glomerular inflammation and is the most common cause of renal failure worldwide. Glomerular fibrin
deposits are observed in severe proliferative and in crescentic forms of human GN, in which the outcome for renal
function is often poor (5). These forms of GN are associated with recruitment of T cells and monocytes (6) and
cross-linking of fibrin both in the glomerular tuft and within
Bowman's space. Fibrin in Bowman's space is chemotactic for macrophages (7). This accumulation of macrophages
and fibrin forms the histological appearance of a crescent
(8, 9).
The glomerulus, being a complex filter, is particularly
susceptible to injury induced by local deposition of fibrin
and other matrix proteins. Defibrination studies have demonstrated that fibrin is an important mediator of glomerular
injury and crescent formation in experimental GN (8, 9).
Local dysregulation of coagulation (10) and fibrinolysis (11)
may contribute to glomerular fibrin deposition. Upregulation of tissue factor has been demonstrated in experimental
(12) and human crescentic GN (13). Dysregulation of the
plasminogen/plasmin system, with decreased glomerular fibrinolytic activity, decreased tissue type plasminogen activator (tPA) and increased plasminogen activator inhibitor-1
(PAI-1) has also been demonstrated in experimental crescentic GN (11) and in human GN (14).
The extent to which local generation of plasmin plays a
protective role in preventing glomerular fibrin deposition
in crescentic GN is not known. Plasmin can cleave crosslinked fibrin, directly degrade matrix proteins (1) and can
activate matrix metalloproteinases (15), suggesting a protective function for plasmin in the glomerulus. Conversely,
plasmin may activate latent transforming growth factor- To evaluate the plasminogen/plasmin system in immune
initiated glomerular injury, proliferative GN was induced in
mice with genetic deficiencies of key molecules of this system. These included mice deficient in plasminogen (Plg Study Design.
Mice deficient in plasminogen, tPA, uPA, both
tPA and uPA, and uPAR were created and characterized as previously described (2, 19, 20). Mice were 48-69 d old and clinically
free of disease at induction of GN. Studies of Plg (16), which promotes matrix deposition (17), enhances
PAI-1 production (17) and down regulates plasminogen
activator production in the kidney (18).
/
)
(19), mice with combined deficiencies of tPA and uPA
(tPA
/
:uPA
/
) (2), mice with single deficiencies of
either tPA (tPA
/
) or urokinase-type plasminogen activator (uPA
/
) (2) and mice deficient in the cell surface receptor for uPA, (uPAR
/
) (20). Mice were compared
with their appropriate genetically unmodified (or wild-type
[WT]) mice in each case. GN was induced by planting an
exogenous Ag, sheep anti-mouse GBM globulin, in the
glomeruli of mice previously sensitized to sheep globulin
(SG) which induced a rapidly progressive, proliferative GN
with renal impairment in WT mice.
/
, tPA
/
,
uPA
/
, uPAR
/
mice and their WT mice used male mice
weighing between 20 and 30 g, except Plg
/
mice, which
weighed between 10.7 and 21.3 g. tPA
/
:uPA
/
mice and
their WT controls were female. WT female mice weighed 16.9 to 20.5 g and tPA
/
:uPA
/
mice weighed 13.7 to 18.8 g.
Assessment of Glomerular Injury. Renal tissue was fixed in Bouin's fixative, embedded in paraffin, cut in 3 µm sections and stained with periodic acid Schiff's stain (PAS) for histological analysis. Glomerular cellularity was determined by counting cell nuclei in at least 20 glomeruli per animal and results expressed as cells per glomerular cross section (cells/gcs). The presence of PAS+ material within glomeruli was scored semiquantitatively in a minimum of 50 glomeruli per mouse as follows: 0 = no deposition of PAS+ material, 1 = up to one third of the cross-sectional area of the glomerulus staining PAS+, 2 = one third to two thirds involvement, 3 = greater than two thirds involvement. Crescent formation (defined as the presence of 3 or more layers of cells in Bowman's space) was assessed (minimum of 50 glomeruli per mouse) and results expressed as a percentage incidence.
Tissue sections (6 µm) were stained to demonstrate macrophages using a three-layer immunoperoxidase technique, as previously described (22). The primary Ab was M1/70 (monoclonal anti-mouse Mac-1; American Type Culture Collection [ATCC], Rockville, MD). Sections of spleen provided a positive control for each animal and protein G-purified rat Ig was substituted for the primary Ab to provide a negative control. A minimum of 20 glomeruli were assessed per mouse and results expressed as cells/gcs. Fibrin immunofluorescence was performed on sections (4 µm) cut from frozen tissue. An IgG fraction of goat anti-mouse fibrin/ fibrinogen serum (Nordic Immunological Laboratories, Drawer, CA) was conjugated to digoxigenin (Boehringer Mannheim, Castle Hill, Australia) and used at a concentration of 35 µg/ml. Binding of the primary Ab was detected with FITC-conjugated sheep anti-digoxigenin Fab fragments (Boehringer Mannheim) at a dilution of 1 in 50. Glomerular fibrin deposition was assessed semiquantitatively (minimum of 30 glomeruli per mouse) using the scoring system described for the assessment of glomerular PAS+ material. Serum creatinine (sCr) was measured by the alkaline picric acid method by autoanalyzer (Cobas Bio, Roche Diagnostics, Basel, Switzerland). As sCr is proportional to weight, values for PlgMeasurement of Circulating Mouse Anti-Sheep Globulin Antibody.
Mouse anti-sheep globulin titers were assessed by ELISA
on serum collected at the end of the experiment. Microtiter plates were coated with 10 µg/ml SG in carbonate/bicarbonate buffer
(pH 9.5), washed, blocked with 1% BSA, then incubated with
dilutions of mouse serum. Bound mouse Ig was detected with
horseradish peroxidase conjugated rabbit anti-mouse Ig (Sigma
Chemical Co.) at a dilution of 1 in 2,000 using 0.1 M 2,2-azinodi-3-ethylbenzthiazoline sulphonate (ABTS; Boehringer Mannheim, Castle Hill, Australia) in 0.02% H2O2 as the substrate. The
absorbance at 405 nm was read on a microtiter plate reader. Serum from each mouse was tested in serial dilutions from 1 in 100 to 1 in 126,000, with serum from six non-immunized mice used
as negative controls.
Statistics.
Results are expressed as the mean ± SEM. Significances of differences were determined by the unpaired t test for
the Plg/
and tPA
/
:uPA
/
mice and their respective WT.
Multiple group comparisons (i.e., tPA
/
, uPA
/
, uPAR
/
and WT mice) were analyzed by ANOVA, followed by Fisher's
protected least significant differences (PLSD) test.
All WT mice developed proliferative GN (Fig. 1 A) with occasional glomeruli exhibiting
crescent formation. Areas of basement membrane thickening
were demonstrated by silver methenamine staining (not
shown) and segmental fibrin deposition was observed within the glomerular tuft by direct immunofluorescence (Fig. 1
B). Glomeruli of both Plg/
and tPA
/
:uPA
/
mice
were more severely affected, with tuft necrosis and relatively hypocellular glomeruli (Fig. 1, C and E). Despite this
relative hypocellularity, there were more macrophages within
glomeruli and significantly increased numbers of glomeruli
exhibiting crescent formation compared to their respective
WT controls (Table 1). Crescents in the Plg
/
and tPA
/
:
uPA
/
animals were larger than those found in WT mice
and contained both cellular elements (Fig. 1, C and E, arrowed), and fibrin (Fig. 1, D and F, arrowed). Deposition of
PAS+ material within glomeruli was significantly increased
(Table 1) indicating more extra cellular matrix deposition in
nephritic Plg
/
and tPA
/
:uPA
/
mice, compared
to WT animals.
|
Of mice deficient in a single plasminogen activator or
uPAR, only tPA/
mice exhibited histologically more
severe disease with increased glomerular hypercellularity
and increased numbers of glomerular macrophages. Deposition of PAS+ material was increased compared to WT
mice (Table 1) but did not reach the extent of the Plg
/
or tPA
/
:uPA
/
mice. Mice deficient in uPA had
fewer macrophages infiltrating glomeruli compared to WT
mice, but no other differences in the histological appearances or quantitative indices of renal injury (Table 1). Nephritic uPAR
/
mice showed similar histological appearances to WT mice (Table 1).
In contrast to the
segmental fibrin deposition observed only within the glomerular tuft in WT mice (Fig. 1 B), Plg/
and tPA
/
:
uPA
/
mice developed extensive glomerular fibrin deposition within the glomerular tuft and in Bowman's space
(Fig. 1, D and F). Semi-quantitative scoring of the extent
of glomerular fibrin deposition confirmed that these differences were statistically significant compared to their WT
controls (Fig. 2). Mice deficient in tPA also showed significant increases in glomerular fibrin deposition over WT
(Fig. 3), though the extent of glomerular fibrin deposition
did not reach that of the Plg
/
or the tPA
/
:uPA
/
mice. There were no significant changes in glomerular fibrin deposition in nephritic uPA
/
and uPAR
/
mice
compared to WT mice.
Effect of Plasminogen and Plasminogen Activator Deficiencies on Renal Function in GN.
The importance of the increased
glomerular deposition of fibrin and other matrix proteins
induced by deficiency of plasminogen activation was demonstrated by the increased impairment of renal function observed in plasminogen or plasminogen activator-deficient mice. Plg/
and tPA
/
:uPA
/
mice developed more
severe renal failure, measured by sCr compared to their appropriate WT groups (Fig. 2). Mice deficient in tPA alone
also developed more renal impairment compared to their
WT control. uPA
/
mice showed a trend towards more
severe renal impairment (sCr P = 0.08 compared to WT).
The severity of renal impairment of nephritic uPAR
/
mice was unchanged from that of WT mice with GN (Fig. 3).
In addition to
the activity of the coagulation and fibrinolytic systems, the
extent of immune response to the disease initiating antigen
is important in determining the severity of injury in this
model of GN. Although deficiencies of the plasminogen/
plasmin system are not known to modulate immune responses, the specific immune response to the nephritogenic
Ag (SG) was quantified in the various groups of mice with
GN by measurement of circulating levels of mouse anti-sheep
globulin antibodies. Ab titers, measured by ELISA at dilutions of sera from 1 in 100 to 1 in 102,400, were not significantly different for any genetically deficient mice compared
with their WT control group, except in Plg/
mice that showed slightly lower absorbance values at the two highest
antibody dilutions. These minor differences are unlikely to
significantly modulate the expression of GN and could not
contribute to the more severe disease seen in these Plg
/
mice.
These studies demonstrate that generation of plasmin
plays a critical role in protecting glomerular structure and
function in rapidly progressive GN. A similar degree of severe glomerular damage was observed with plasminogen
deficiency and combined deficiency of tPA and uPA, suggesting that these two plasminogen activators contribute
the major plasminogen activating activity in GN. This supports the earlier observation that tPA and uPA are the only two physiologically significant plasminogen activators in
mice (19). Mice deficient in tPA alone also developed
more severe renal injury than WT mice, but not to the
extent observed in combined PA or the Plg-deficient mice.
More severe GN did not develop in uPA/
or uPAR
/
mice.
In the initial characterization of mice with combined deficiencies of tPA and uPA, minor spontaneous renal fibrin
deposition was reported in a minority of animals beyond
the age of 2 to 3 mo (2). However, in this study disease was
induced in tPA/
:uPA
/
mice that were younger than 2 mo old to avoid any possibility that spontaneous glomerular
fibrin deposition may significantly influence the results. In
the original description of Plg
/
mice, dilated renal
pelvises had been noted in four mice, without fibrin deposition (19). None of the Plg
/
mice in the current study had macroscopic or microscopic evidence of pelvicalyceal
dilatation or obstruction.
There was a significantly increased propensity to form
crescents in glomeruli of mice with plasminogen or combined plasminogen activator deficiency. Crescents are the
result of accumulation of fibrin and macrophages, together
with proliferation of glomerular epithelial cells in Bowman's space. They are indicative of severe glomerular injury
and a poor outcome for renal function. Fibrin has previously been shown to be an important chemotactic stimulus for accumulation of macrophages in Bowman's space and
crescent formation (7). The enhanced crescent formation
and macrophage infiltration in Plg/
and tPA
/
:
uPA
/
mice is consistent with the observation that enhanced glomerular fibrin accumulation occurring in the
absence of local plasmin generation provides a greater stimulus for inflammatory cell recruitment and crescent formation. The more intense inflammatory reaction resulted in
necrosis of the glomerular tuft and hypocellular glomeruli.
tPA is the predominant PA within mouse (23), rabbit (11) and human (24, 25) glomeruli and is produced mainly by glomerular endothelial cells. The major site of uPA production within the murine kidney is the tubular epithelium (23), although human glomerular epithelial cells in culture also produce uPA (24, 26) and uPA immunofunctional activity has been demonstrated in rabbit (11) and human glomeruli (25). uPA has been proposed to have a role in maintenance of tubular patency (23).
Mice deficient in tPA alone showed significantly increased
renal failure, glomerular hypercellularity, glomerular macrophage numbers, glomerular fibrin deposition and deposition
of PAS+ material within glomeruli. They also demonstrated
a trend towards increased crescent formation. Glomerular
capillary thrombosis was minimal in nephritic tPA/
mice
and their glomeruli were hypercellular. However, the severity of injury in these mice was not sufficient to cause necrosis of the glomerular tuft.
Mice deficient in uPA alone did not show significant differences in histological indices of disease from WT mice,
but did show a trend towards increased renal impairment.
The significance of this result is unclear, but the extensive
expression of uPA in renal tubular epithelial cells of normal
mice suggests that tubular dysfunction may have contributed to any increased renal impairment in uPA/
mice.
The demonstration of significantly reduced glomerular macrophage infiltration in uPA
/
mice supports the hypothesis that uPA assists migration of macrophages to sites of inflammatory injury.
The increased renal impairment, fibrin deposition and
other histological indices of disease in the tPA/
mice
suggest a more important role for tPA in protecting glomeruli against inflammatory injury, but do not exclude a role
for uPA. However, the more severe glomerular injury in
mice deficient in both plasminogen activators implies that
the effects of uPA deficiency in glomerular inflammation become evident in the absence of tPA.
Deficiency of the uPA receptor did not significantly modify the histological or functional manifestations of GN. The observation that glomeruli of uPAR-deficient mice contained similar numbers of macrophages to WT mice suggests that unlike uPA, uPAR is not important in macrophage infiltration into nephritic glomeruli. In normal mice, uPAR mRNA is expressed only at low levels in the kidney, in contrast to the abundant renal expression of uPA mRNA (27). Sites of uPA expression in the kidney do not correspond well to its receptor (27), suggesting that in the kidney uPA can act independently of uPAR. Although a prominent role for uPA and uPAR could not be demonstrated in this short term model of renal injury, this does not preclude a significant role for these proteins in chronic renal injury, where prolonged accumulation of matrix proteins leads to renal scarring and glomerulosclerosis.
In summary, these studies demonstrate an important protective role for plasminogen and plasminogen activators in acute inflammatory renal injury associated with crescentic GN. They are consistent with previous observations that tPA and uPA appear to be the major plasminogen activators in vivo but also suggest that the major protective role in acute inflammatory glomerular injury is attributable to tPA. uPA facilitates recruitment of macrophages into nephritic glomeruli. The uPA receptor does not appear to play any significant role in protecting the glomerulus in GN. Therapies aimed at enhancing or replacing fibrinolytic activity in crescentic GN may be of benefit in human disease.
Address correspondence to Dr. A. Richard Kitching, Monash University Department of Medicine, Monash Medical Centre, 246 Clayton Road, Clayton 3168, Victoria, Australia.
Received for publication 2 August 1996 and in revised form 24 October 1996.
The expert assistance of L. Moons, E. Simic, J. Tsoupas, P. Davenport, B. Hermans, I. Cornelissen and S. Jansens is acknowledged.This work was supported by grants from the National Health and Medical Research Council (NH&MRC) of Australia, the Flemish Interuniversity Institute of Flanders and Human Frontiers in Science Program.
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