Plasminogen Activator Inhibitor-1 Is a Significant Determinant of Renal Injury in Experimental Crescentic Glomerulonephritis
A. Richard Kitching*,,
Yao Z. Kong*,
Xiao Ru Huang*,
Piers Davenport*,
Kristy L. Edgtton*,
Peter Carmeliet,
Stephen R. Holdsworth* and
Peter G. Tipping*
*Centre for Inflammatory Diseases, Monash University, Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia; and Centre for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.
Correspondence to Dr. Peter G. Tipping, Monash University Department of Medicine, Monash Medical Centre, 246 Clayton Road Clayton, Victoria 3168, Australia. Phone: 61-3-9594-5547; Fax: 61-3-9594-6495;
ABSTRACT. Crescentic glomerulonephritis is characterized byglomerular fibrin deposition, and experimental crescentic glomerulonephritishas been shown to be fibrin-dependent. Net fibrin depositionis a balance between activation of the coagulation system causingglomerular fibrin deposition and fibrin removal by the plasminogen-plasmin(fibrinolytic) system. Plasminogen activator inhibitor-1 (PAI-1)inhibits fibrinolysis by inhibiting plasminogen activators andhas effects on leukocyte recruitment and matrix deposition.To test the hypothesis that the presence of PAI-1 and its levelswere a determinant of injury in crescentic glomerulonephritis,accelerated anti-glomerular basement membrane glomerulonephritiswas induced in mice genetically deficient in PAI-1 (PAI-1 -/-),PAI-1 heterozygotes (PAI-1 +/-), and mice engineered to overexpressPAI-1 (PAI-1 tg). Compared with strain-matched genetically normalanimals, PAI-1 -/- mice with glomerulonephritis developed fewerglomerular crescents, less glomerular fibrin deposition, fewerinfiltrating leukocytes, and less renal collagen accumulationat day 14 of disease. The reduction in disease persisted atday 28, when injury had become more established. In contrast,mice overexpressing the PAI-1 gene (PAI-1 tg), that have basalplasma and renal PAI-1 levels several times, normal developedincreased glomerular crescent formation, more glomerular fibrindeposition, increased numbers of infiltrating leukocytes, andmore renal collagen at both time points. These studies demonstratethat PAI-1 is a determinant of glomerular fibrin depositionand renal injury in crescentic glomerulonephritis. E-mail: peter.tipping@med.monash.edu.au
Crescentic glomerulonephritis (GN) is characterized by glomerularfibrin deposition (1,2). Glomerular fibrin deposition is a featureof rapidly progressive GN in both humans and in experimentalmodels (3,4) In experimental systems, this lesion is fibrin-dependent(5,6). Net glomerular fibrin deposition is a balance betweenactivation of the coagulation system and fibrin deposition andfibrin removal by the plasminogen-plasmin system (7,8). Plasminogenactivator inhibitor-1 (PAI-1) is a serine protease inhibitorthat inhibits the activity of both tissue type plasminogen activator(tPA - the predominant glomerular plasminogen activator (9))and urokinase type PA (uPA) (reviewed in references 10 and 11).PAI-1 is upregulated early in the course of experimental immunerenal injury (12,13). Inhibition of tPA and uPA by PAI-1 leadsto multiple events that favor deposition of fibrin and othermatrix proteins, by inhibiting plasmin generation and the directeffects of uPA. PAI-1 has other effects on cell migration andleukocytes that seem to favor recruitment of leukocytes to inflammatorylesions (11). There is some evidence, including studies of invivo fibrotic interstitial renal injury (14), that PAI-1 hasa pathogenetic role in renal disease. Recent reviews have highlightedboth the pathogenetic potential for PAI-1 in renal disease andits potential role as a therapeutic target (10,11).
Functional studies of the role of fibrin in crescentic GN haveshown that this fibrin deposition is an important mediator ofinjury (4–6,15). The net amount of fibrin accumulationin glomeruli results from pathogenetic procoagulant effectsdriven by tissue factor (7,16) and the protective effects ofthe plasminogen plasmin system (8,12). Studies in mice deficientin components of this system have demonstrated a protectiverole for endogenous plasmin (generated by the conversion ofplasminogen to plasmin by tPA) in experimental crescentic GN(8). Increased glomerular PAI-1 expression has been found inboth human and experimental crescentic GN (12,17–19).Given these findings, it is logical that molecules that inhibitplasmin generation, such as PAI-1, would be pathogenetic increscentic GN. However, this hypothesis has not been testedin vivo.
These studies address the hypothesis that in fibrin-associatedimmune glomerular injury, PAI-1 is an important determinantof injury, crescent formation, and matrix accumulation. Theyuse mice that have been genetically manipulated to be PAI-1–deficient(PAI-1 -/- mice) or PAI-1 overexpressing (PAI-1 tg mice) inwhich experimental crescentic GN, a fibrin associated form ofimmune glomerular injury, had been induced. In addition to studyingdisease at 14 d, experimental crescentic GN was studied at 28d to determine whether any effect of PAI-1 on fibrin/matrixdeposition and injury could be overcome by other inhibitorsof plasmin.
Experimental Design
Mice with a genetic deletion of PAI-1 (20,21) on a C57BL/6 x129/SvJ background, backcrossed once onto a C57BL/6 background(75% C57BL/6, 25% 129/SvJ) were bred in a specific pathogen-freefacility (Monash Medical Center, Melbourne, Australia). Miceof an identical genetic background (i.e., 75% C57BL/6, 25% 129/SvJ)bred from the backcross were used as controls. PAI-1 +/- micewere generated by interbreeding PAI-1 -/- and PAI-1 +/+ mice.PAI-1 transgenic mice on a pure C57BL/6 background expressinga murine PAI-1 minigene under a CMV promoter were generouslyprovided by Dr. D. Ginsburg (Howard Hughes Medical Institute,University of Michigan Medical Center, Ann Arbor, Michigan).(22) PAI-1 tg mice were bred in a specific pathogen-free facility(Monash Medical Center). Genetically normal pure C57BL/6 micewere used as controls. Male mice aged 8 to 12 wk were used forexperiments. Anti-mouse glomerular basement membrane (GBM) globulinwas prepared as described previously (23) Mice were sensitizedby subcutaneous injection of a total of 2 mg of sheep globulinin 100 µl of Freund complete adjuvant in divided dosesin each flank 10 d before challenge with 5 mg of sheep anti-mouseGBM globulin. Renal injury was studied at two time points: 14d and 28 d after challenge with sheep anti-mouse GBM globulin.Results are expressed as the mean ± SEM. The significanceof differences between groups was determined by ANOVA, followedby Bonferroni Multiple Comparison Test for paired comparisons(GraphPad Prism; GraphPad Software Inc., San Diego, CA). Forstudies involving PAI-1 tg mice (two groups only) the unpairedt test was used.
For studies using mice genetically deficient in PAI-1, the followinggroups of mice were studied:
PAI-1 +/+ mice: baseline (n = 6), GN day 14 (n = 6), GN day28 (n = 6)
PAI-1 +/- mice: baseline (n = 6), GN day 14 (n= 6), GN day28 (n = 6)
PAI-1 -/- mice: baseline (n = 7),GN day 14 (n = 7), GN day28 (n = 7)
For studies using mice genetically engineered to overexpressPAI-1, the following groups of mice were studied:
C57BL/6 mice: baseline (n = 8), GN day 14 (n = 8), GN day 28(n = 8)
PAI-1 tg mice (C57BL/6): baseline (n = 6), GN day14 (n = 6),GN day 28 (n = 6)
Assessment of PAI-1 Gentoype and Plasma and Renal PAI-1 Levels
PCR based protocols were used to determine the genotype of PAI-1+/+, PAI-1 +/-, PAI-1 -/-, and PAI-1 tg mice. The PAI-1 transgenewas detected as described previously (22). PAI-1 -/-, PAI-1+/-, and PAI-1 +/+ mice were genotyped by using primers forthe wild-type PAI-1 gene (22) and by detecting the presenceof the neomycin resistance gene as a marker for the mutant allele(20). The absence of the PAI-1 was confirmed by measurementof PAI-1 protein in plasma via ELISA using monoclonal antibodiesgenerated by immunizing genetically deficient mice as describedpreviously (24). Results are expressed as ng of PAI-1 proteinper ml of plasma. For measurement of PAI-1 in renal tissue byELISA, tissue from mice was homogenized and extracted as describedpreviously by Lijnen et al. (25) and results expressed as pgof PAI-1 per mg of wet weight.
Assessment of Glomerular Crescent Formation and Glomerular Fibrin Deposition
Kidney tissue was fixed in Bouin fixative and embedded in paraffin,and 3-µm tissue sections were cut and stained with periodicacid-Schiff (PAS). A glomerulus was considered to exhibit crescentformation if two or more layers of cells were observed in Bowman’sspace. A minimum of 50 glomeruli was assessed to determine thecrescent score for each animal. For detection of fibrin in glomeruli,tissue was embedded in Optimal Cutting Temperature Compound,frozen in liquid nitrogen, and stored at -70°C. Immunofluorescencewas performed on 4-µm cryostat cut tissue. Glomerularfibrin deposition was assessed on a minimum of 30 glomeruliper mouse using FITC-goat anti-rabbit fibrin/fibrinogen serum(Nordic Immunological Laboratories, Berks, UK), which cross-reactswith mouse fibrin at a dilution of 1:100 and scored semiquantitatively(0 to 3+) using two different scales. First, the proportionof glomeruli staining positive for fibrin in each mouse wasassessed. Second, the overall intensity of fluorescence presentin each mouse was scored.
Glomerular Leukocyte Accumulation
Kidney tissue was fixed in periodate lysine paraformaldehyde(PLP) for 4 h, washed in 7% sucrose solution, and then frozenin liquid nitrogen. Tissue sections (6 µm) were stainedto demonstrate CD3+ cells and macrophages using a three-layerimmunoperoxidase technique, as described previously (26,27).The primary monoclonal antibodies were KT3, anti-mouse CD3,which recognizes T cells (American Type Culture Collection [ATCC],Manassas, VA), and M1/70, anti-CD11b, which recognizes macrophagesand neutrophils (ATCC). A minimum of 20 glomeruli were assessedper animal, and results were expressed as cells per glomerularcross section (c/gcs).
Assessment of Proteinuria and Serum Creatinine
Urinary protein concentrations were determined by the Bradfordmethod on timed urine collections. Urine was collected for 24h at baseline and over the last 24 h of an experiment. Serumcreatinine concentrations at the completion of experiments (day14 or day 28) were measured by the alkaline picric acid methodusing an autoanalyser.
Renal Collagen and Matrix Accumulation
Renal collagen content was assessed by determining total hydroxyprolineaccording to the method of Bergam and Loxley (28). Samples werehydrolyzed in 6 N HCL by incubation at 110°C overnight.The hydrolysate was neutralized with 2.5 M NaOH. Hydrolysatesin isopropanol were oxidized by chloramine T then mixed withp-dimethylsaminonbenzaldehyde (25 min, 60°C), and the absorbancewas measured at 558 nm. Total collagen was calculated usingthe assumption that collagen contains 12.7% hydroxyproline byweight. Results were expressed as µg/mg kidney wet weight.
PAI-1 Protein Levels are Undetectable in PAI-1 -/- Mice and Increased in PAI-1 tg Mice
Plasma PAI-1 levels in normal (without GN) PAI-1 +/+ mice (C57BL/6x 129Sv/J background) were 9.7 ± 1.5 ng/ml (Table 1).PAI-1 protein was undetectable in all PAI-1 -/- mice. PAI-1+/- mice had intermediate levels of PAI-1 protein (reduced to30 to 40% of normal). Normal C57BL/6 mice had PAI-1 levels fallingbetween those of PAI-1 +/- and PAI-1 +/+ mice. Mice engineeredto genetically overexpress PAI-1 (PAI-1 tg mice) produced significantlymore PAI-1 than all other mice (72 ± 8.1 ng/ml).
Table 1. Plasma levels of PAI-1 protein measured by ELISA in mice without glomerulonephritis
Glomerulonephritis is Less Severe in Mice Lacking PAI-1 Endogenous PAI-1 Mediates Glomerular Crescent Formation and Fibrin Deposition.
Genetically normal PAI-1 +/+ mice developed proliferative GNwith glomerular crescent formation and fibrin deposition 14d after challenge with sheep anti-mouse GBM globulin (Figures 1, B and E;Figure 2). Glomerular crescent formation and fibrindeposition persisted to day 28. PAI-1 -/- mice were significantlyprotected from glomerular crescent formation at both the day14 and day 28 time points (Figures 1C and 2A). Renal PAI-1 proteinlevels progressively increased in PAI-1+/+ mice but remainedundetectable in PAI-1-/- mice (Table 2). A modest reductionin crescent formation was observed in PAI-1 +/- mice at day14, but not at day 28. Fibrin deposition was detected in mostglomeruli of PAI-1 +/+ mice, mainly in the glomerular tuft.However, fibrin deposition was absent in over half of glomeruliin PAI-1 -/- mice (Figures 1F and 2B). At the later time point(day 28), there had been no progressive increase in glomeruliaffected in PAI-1 -/- mice. In addition to there being fewerglomeruli in which fibrin was detected, overall intensity offluorescence was decreased in PAI-1–deficient mice (0to 3+, per mouse) (day 14: PAI-1 +/+ 2.8 ± 0.2, PAI-1-/- 1.4 ± 0.2; day 28: PAI-1 +/+ 1.8 ± 0.3, PAI-1-/- mice all a score of 1).
Figure 1. Glomerular injury and fibrin deposition in plasminogen activator inhibitor-1 (PAI-1) +/+ and PAI-1 -/- mice with glomerular nephritis (GN) 14 d after the initiation of injury. (A) Glomerulus from a PAI-1 +/+ mouse without GN. (B) PAI-1 +/+ mice developed proliferative crescentic glomerular injury (mean % glomeruli affected, see Figure 2A). (C) PAI-1 -/- mice developed proliferative GN that is significantly less severe than PAI-1 +/+ mice with only occasional crescent formation. (D) Negative immunofluorescence for fibrin in a glomerulus from a PAI-1 +/+ mouse without GN. (E) Significant fibrin deposition in the glomerular tuft of a PAI-1 +/+ mouse with GN. (F) Reduced fibrin deposition in a glomerulus of a PAI-1 -/- mouse with GN. (A through C, periodic acid-Schiff [PAS] stain, high power view; D through F, immunofluorescence, high power).
Figure 2. Glomerular crescent formation and fibrin deposition in PAI-1 +/+, PAI-1 +/-, and PAI-1 -/- mice with GN at 14 and 28 d. Open bars represent PAI-1 +/+ mice, gray bars represent PAI-1 +/- mice, and black bars represent PAI-1 -/- mice. Crescent formation (A) and fibrin deposition (B, expressed as the proportion of glomeruli with immunofluorescent fibrin deposits present) are markedly reduced in mice that are genetically deficient in PAI-1. * P < 0.05 versus PAI-1 +/+ mice, ** P < 0.001 versus either PAI-1 +/+ or +/- mice at the same timepoint (ANOVA).
Table 2. Renal PAI-1 protein levels measured by ELISA in PAI-1 intact and deficient mice with GN
Leukocyte Recruitment Is Diminished in the Absence of PAI-1.
CD3+ T cells and macrophages were detected in glomeruli of geneticallynormal PAI-1 +/+ mice (Figure 3). While leukocyte numbers wereunchanged in PAI-1 heterozygotes, complete absence of PAI-1resulted in a substantial reduction in both T cells and macrophagesat both time points.
Figure 3. Glomerular leukocyte accumulation in PAI-1 +/+, PAI-1 +/-, and PAI-1 -/- mice with GN at 14 and 28 d. Open bars represent PAI-1 +/+ mice, gray bars represent PAI-1 +/- mice, and black bars represent PAI-1 -/- mice. T cells (A) and CD11b+ cells (B) are markedly reduced in glomeruli of mice that are genetically deficient in PAI-1. Dotted lines represent values for mice without GN. ** P < 0.001 versus either PAI-1 +/+ or +/- mice at the same time point (ANOVA).
Functional Indices of Renal Injury in the Absence of PAI-1.
Baseline urinary protein and renal function (measured by serumcreatinine) were similar in the three experimental groups (urinaryprotein: 0.4 ± 0.1 mg/d in all groups; serum creatinine:PAI-1 +/+ 17 ± 0.4, PAI-1 +/- 17 ± 0.5, PAI-1-/- 19 ± 0.4 µmol/L). PAI-1 +/+ and PAI-1 +/- micehad developed significant proteinuria and renal impairment byday 14 of disease. Urinary protein excretion in PAI-1 -/- micewith GN was significantly lower than the proteinuria observedin either PAI-1 +/+ or PAI-1 +/- mice at day 14 and lower thanproteinuria in PAI-1 +/- mice at day 28 (Figure 4A). Serum creatininelevels in PAI-1 -/- mice at either day 14 or day 28 were notdifferent from baseline values (Figure 4B).
Figure 4. Functional renal injury in PAI-1+/+, PAI-1 +/-, and PAI-1 -/- mice with GN at 14 and 28 d. Open bars represent PAI-1 +/+ mice, gray bars represent PAI-1 +/- mice, and black bars represent PAI-1 -/- mice. (A) Proteinuria is reduced in PAI-1 -/- mice, though this reduction, compared with PAI-1 +/+ mice, reached statistical significance only at 14 d. (B) There was a trend to decreased serum creatinine in PAI-1 -/- mice. The dotted line represents the mean urinary protein excretion (A) and serum creatinine (B) of mice without GN. There was no difference in these baseline values between groups. * P < 0.05 versus PAI-1 +/+ mice and < 0.001 versus PAI-1 +/- mice, ** P < 0.01 versus PAI-1 +/- mice. *** P < 0.001 versus PAI-1 -/- and +/+ mice.
Renal Collagen Content in GN in the Absence of Endogenous PAI-1.
Genetic deletion of PAI-1 did not affect baseline renal collagenlevels (Table 3). By day 14 of disease, PAI-1 +/+ mice had developeda 100% increase in renal collagen content, with levels remainingelevated at day 28. Renal collagen content in PAI-1 -/- micewas significantly less that that in PAI-1 +/+ mice at day 14,but this decrease failed to reach statistical significance atday 28. Collagen accumulation in PAI-1 +/- mice was more thanin PAI-1 -/- mice but less than PAI-1 +/+ mice at day 14 ofGN.
Table 3. Renal collagen content in PAI-1 intact and deficient mice with GN
Glomerulonephritis is More Severe in Mice Overexpressing PAI-1 Overexpression of PAI-1 Enhances Glomerular Crescent Formation and Glomerular Fibrin Deposition.
Normal C57BL/6 mice developed proliferative and crescentic GN14 d after challenge with sheep anti-mouse GBM globulin, withglomerular fibrin deposition (Figures 5A, 5C, and 6). Overall,disease in these mice was marginally less than disease in PAI-1+/+ mice on a mixed 75% C57BL/6 x 25% 129Sv/J background. Crescentformation and glomerular fibrin deposition persisted at day28. Renal PAI-1 protein was increased in PAI-1 tg mice, mostevident at day 14 (Table 4). At day 14, crescentic GN was moresevere in PAI-1 tg mice compared with normal C57BL/6 mice (Figures 5B, 5D, and 6).An increased proportion of glomeruli from PAI-1tg mice was affected by crescent formation and more glomeruliexhibited fibrin deposition. This increased severity of diseasepersisted at day 28. Overall intensity of fibrin depositionwas also increased in PAI-1 tg mice. C57BL/6 mice with GN hadmoderate fluorescence intensity for fibrin (0 to 3+ [mean ±SEM]: day 14, 1.5 ± 0.2; day 28, 0.6 ± 0.1). Sectionsfrom all PAI-1 tg mice developed strong immunofluorescence forfibrin (all 3+) at day 14, which was still moderate in intensityat day 28 (1.6 ± 0.3).
Figure 5. Glomerular injury and fibrin deposition in genetically normal C57BL/6 mice and C57Bl/6 PAI-1 transgenic (PAI-1 tg) mice with GN 14 d after the initiation of injury. (A) Significant glomerular injury with some crescent formation was present in C57BL/6 mice with (mean % glomeruli affected see Figure 6A). (B) C57BL/6 PAI-1 tg mice developed accelerated crescentic GN compared with genetically normal mice with more frequent crescent formation. (C) Fibrin deposition in the glomerular tuft of a C57BL/6 mouse with GN. (D) Increased glomerular fibrin deposition in a glomerular from a PAI-1 tg mouse with GN. (A and B, PAS stain, high power view; C and D, immunofluorescence, high power, photographed at the same exposure).
Figure 6. Glomerular crescent formation and fibrin deposition in genetically normal C57BL/6 mice and PAI-1 overexpressing (PAI-1 tg) mice with GN at 14 and 28 d. Open bars represent genetically normal C57BL/6 mice, and gray bars represent PAI-1 tg C57BL/6 mice. Crescent formation (A) and fibrin deposition (B, expressed as the proportion of glomeruli with immunofluorescent fibrin deposits present) are increased in mice that genetically overexpress PAI-1. * P < 0.005, ** P < 0.001 versus C57BL/6 mice at the same time point (unpaired t test).
Leukocyte Recruitment Is Enhanced in PAI-1 tg Mice.
Mice that overexpressed PAI-1 demonstrated increased recruitmentof T cells and CD11b+ leukocytes into glomeruli compared withgenetically normal C57BL/6 mice with GN (Figure 7). This increasedleukocyte influx in PAI-1 tg mice persisted at day 28 of disease.
Figure 7. Glomerular leukocyte accumulation in genetically normal C57BL/6 mice and PAI-1 overexpressing (PAI-1 tg) mice with GN at 14 and 28 d. Open bars represent genetically normal C57BL/6 mice, gray bars represent PAI-1 tg C57BL/6 mice. T cells (A) and CD11b+ cells (B) are present in increased numbers in glomeruli of mice that genetically overexpress PAI-1. Dotted lines represent values for mice without GN. * P < 0.005, ** P < 0.001 versus C57BL/6 mice at the same timepoint (unpaired t test).
Functional Renal Injury Is Enhanced by PAI-1 Overexpression.
C57BL/6 mice with GN developed significant proteinuria, measuredat both day 14 and day 28, with only minor renal impairment,consistent with the degree of glomerular crescent formationin these mice (Figure 8). As with glomerular crescent, formation,fibrin deposition, and leukocyte recruitment, PAI-1 tg micewith GN developed increased urinary protein excretion and renalimpairment, evident at both day 14 and day 28.
Figure 8. Functional renal injury in genetically normal C57BL/6 mice and PAI-1 overexpressing (PAI-1 tg) C57BL/6 mice with GN at 14 and 28 d. (A) Proteinuria is increased in PAI-1 tg mice. (B) In PAI-1 tg mice, serum creatinine values were significantly increased compared with normal values and C57BL/6 mice with GN. The dotted line represents the mean urinary protein excretion (A) and serum creatinine (B) of mice without GN. There was no difference in these baseline values between groups. * P < 0.005, ** P < 0.001 versus C57BL/6 mice at the same time point (unpaired t test).
Renal Collagen Accumulation Is Accelerated in PAI-1 tg Mice with GN.
Normal C57BL/6 mice and PAI-1 tg mice had similar baseline renalcollagen (Table 5), indicating that transgenic overexpressionof PAI-1 in the absence of pathologic stimuli does not leadto increased renal collagen content. Compared with C57BL/6 mice,PAI-1 tg mice developed increased renal collagen accumulationat both 14 and 28 d.
PAI-1 has major potential pathogenetic effects in inflammatoryrenal disease, potentially acting on several different mediatorsystems (10,11). By inhibiting the activity of plasminogen activatorsand therefore the generation of plasmin, it can inhibit fibrinremoval, resulting in enhanced fibrin accumulation. In additionto its capacity to enhance net fibrin deposition, PAI-1 hasthe capacity to contribute to the accumulation of collagensand other matrix proteins seen in progressive renal disease.PAI-1 limits conversion of plasminogen to plasmin, the activeenzyme that may activate matrix metalloproteinases and act directlyon matrix to limit collagen matrix accumulation. The increasedPAI-1 levels observed in GN may result in increased leukocyterecruitment in GN both directly (14) and via the chemotacticeffects of fibrin (29).
The alterations in glomerular crescent formation fibrin depositionand leukocyte accumulation in PAI-1 -/- and PAI-1 tg mice demonstratean important role for PAI-1 in experimental crescentic GN. Findingsof alterations in renal collagen content in PAI-1 -/- and PAI-1tg mice support recent studies implicating PAI-1 in renal fibrosis(14). Collectively, the current studies suggest that PAI-1 isimportant in determining the outcome of renal injury. They supportthe hypothesis that inhibition of PAI-1 is a potential strategyin the therapy of both rapidly progressive GN and other progressiverenal diseases.
In the first section of this study, mice genetically deficientin PAI-1 were significantly protected from renal injury in experimentalcrescentic GN compared with genetically matched PAI-1 +/+ miceon a C57BL/6 x 129Sv/J background. The relatively mild injuryin PAI-1 -/- mice did not show a significant "catch up" effectwhen anti-GBM GN was prolonged to 28 d. The protection affordedPAI-1 -/- mice was most significant in crescent formation, fibrindeposition, leukocyte recruitment, and the accumulation of collagen.A secondary finding in these studies was that PAI-1 +/- mice,(carrying one normal allele and one disrupted allele) showeda modest degree of protection in only some parameters of disease,more evident at day 14. However, no protection was apparentat 28 d. These PAI-1 +/- mice had baseline PAI-1 plasma levelsless than half of those of PAI-1 +/+ mice. Assessment of functionalplasma PAI-1 activity in PAI-1 +/- mice after endotoxin stimulationhas shown significant increases, but not to levels seen in PAI-1+/+ mice (20). In the current studies, renal PAI-1 levels inPAI-1 +/- mice were similar to those in PAI-1 +/+ mice at day14. The relative contributions of plasma and tissue derivedPAI-1 in immune renal injury remain unclear.
The second section of these studies used mice that are transgenicfor PAI-1 and produce several times more PAI-1 than geneticallynormal C57BL/6 mice. Control mice in these studies also developedcrescentic GN, though their degree of injury was less that thePAI-1+/+ mice. There are many potential contributors to thevariation in pattern and severity of injury in experimentalGN. Several studies have shown that the genetic background andthe nature of the T cell response are important (30–32).For this reason, the current studies are presented and consideredas two separate studies with separate strain-matched controlgroups. However, it is interesting to note that basal PAI-1levels in C57BL/6 mice were less than those in C57BL/6 x 129Sv/JPAI-1 +/+ mice (plasma and renal). C57BL/6 PAI-1 tg mice withincreased renal PAI-1 levels developed accelerated disease comparedwith C57BL/6 mice when measured by a variety of parameters,including crescent formation, fibrin deposition, glomerularleukocyte accumulation, proteinuria, renal impairment, and renalcollagen accumulation at both day 14 and day 28.
This form of GN is fibrin-mediated, and fibrin is a major substrateof plasmin. Therefore many of the effects observed in this studyare likely to be mediated by the effects of PAI-1 on net fibrinaccumulation in the glomerulus. Fibrin is chemotactic for leukocytes(29), and mice deficient in fibrinogen show fewer leukocytesin glomeruli in this model (6). However, the plasminogen-plasminsystem has multiple potential roles in leukocyte accumulationin GN. Plasmin itself can be chemoattractant for leukocytes(33) and in obstructive uropathy (presumably not mediated byfibrin) mice deficient in plasminogen showed decreased leukocyterecruitment (34). In contrast, in fibrin-mediated GN, plasminogen-deficientmice showed a marked increase in fibrin deposition and an increasein glomerular leukocytes (8), while uPA -/- mice showed increasedleukocytes without increased fibrin deposition (8). In obstructiveuropathy, PAI-1 -/- mice have fewer leukocytes; in the samestudy, PAI-1 was chemotactic for macrophages ex vivo (14). Theexact roles of fibrin, plasmin, and PAI-1 leukocyte recruitmentin the kidney and the mechanisms by which they exert their effectsremain to be fully elucidated.
PAI-1 is a major inhibitor of plasmin via its effects on tPAand uPA. However, other inhibitors of plasmin such as 2–antiplasminhave potential roles in promoting net glomerular fibrin depositionand injury. Studies in mice deficient in 2–antiplasmindo not support a prominent pathogenetic role for this moleculein immune glomerular injury (35). These findings, together withthe current and previous studies (8), suggest that in immuneglomerular injury, PAI-1, and tPA (as well as plasminogen) arethe critical components of the plasminogen–plasmin systemthat determine net glomerular fibrin deposition.
The results of the current studies seem at first glance to bediscordant from those of Dewerchin et al. (36), who found thatrenal fibrin deposition was not significantly reduced in theabsence of PAI-1. However, in the model of acute LPS inducedrenal injury used in those studies, fibrin deposition was extraglomerular.In the model used in our current studies, fibrin depositionis predominantly glomerular. Analysis of renal fibrinolyticactivity after endotoxin injection, as well as renal tPA anduPA activity (by Dewerchin et al. (36)), showed increased renalfibrinolytic activity in PAI-1 -/- mice and preservation ofrenal tPA activity in PAI-1-/- mice (compared with PAI-1 +/+mice, when renal tPA activity fell to undetectable levels).These results are consistent with PAI-1 having an importantrole in protective fibrinolytic activity in murine "anti-GBMGN" as (1) tPA is the major PA in the glomerulus (9); (2) wehave demonstrated tPA to be the more important protective PAin glomerular fibrin deposition in murine crescentic GN (8);and (3) PAI-1-/- mice have preserved renal tPA activity andincreased total renal fibrinolytic activity (36).
The current studies do not address the relative importance ofthe potential sources of PAI-1 in crescentic GN. PAI-1 is presentin plasma and is produced by a variety of renal cells, includingmesangial cells (37,38), endothelial cells (38), and tubularcells (39). It is likely that both tissue-derived and plasma-derivedPAI-1 are important in this disease, though it is probable thattubular cell–derived PAI-1 is not particularly importantin the glomerular fibrin deposition observed in experimentalanti-GBM GN.
There is evidence in human GN that expression of PAI-1 may helpdetermine disease severity. Lupus nephritis, WHO classes IIIand IV, is more severe in humans homozygous for the PAI-1 5'gene promoter 4G polymorphism (4G/4G) (40), which, comparedwith the 5G/5G phenotype, results in increased PAI-1 expression,particularly in response to inflammatory stimuli. Our experimentaldata that disease severity is increased in PAI-1 tg mice anddecreased in PAI-1-/- mice (both strain-matched) support thesehuman observational studies. In summary, the current studiesdemonstrate that PAI-1 is an important determinant of crescentformation, injury, fibrin deposition. and collagen accumulationin experimental crescentic GN.
Acknowledgments
Dr. Ginsburg is thanked for providing the PAI-1 tg mice bredand used in these studies. The assistance of Ms. Janelle Sharkeyand Ms. Alice Wright is acknowledged. These studies were supportedby grants from the National Health and Medical Research Councilof Australia (NH&MRC), the FWO, the Interuniversitary AttractionPoles, the Research Fund K.U.Leuven, and European Union.
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Received for publication October 2, 2002.
Accepted for publication February 15, 2003.
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Abstract
Introduction
2–antiplasmin have potential roles in promoting net glomerular fibrin deposition and injury. Studies in mice deficient in 
