Background

Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis.

Methods

To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO.

Results

Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor- and procollagens 1(I) and 1(III).

Conclusions

These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.

METHODS

Experimental design

The experiments were performed on PAI-1 -/- and +/+ male mice with an identical genetic background (87.5% C57BL6; 12.5% 129SV). Breeding pairs of mice were obtained from Dr. P. Carmeliet (Leuven, Belgium)¹⁷. The genotype was confirmed by Southern blot analysis of ear pinna DNA. Left kidney UUO or sham surgery was performed under general anesthesia when the mice reached an average age of 12.5 weeks. Four groups of PAI-1 +/+ and four groups of PAI-1 -/- mice (N = 3 to 6 per group) were studied: sham surgery, three-day (3D) UUO, seven-day (7D) UUO, and 14-day (14D) UUO. To mark proliferating cells, 5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co., Saint Louis, MO, USA) was injected intraperitoneally (50 g/g body weight) 24 hours prior to the sacrifice. Animals were killed by exsanguination under general anesthesia with isoflurane. The obstructed or sham-operated left kidney was immediately decapsulated, weighed, and cut into several pieces that were stored at -80°C for subsequent studies, or they were fixed in 10% neutral-buffered formalin and embedded in paraffin for histological analysis. All procedures were performed in compliance with guidelines established by the National Research Council Guide for the Care and Use of Laboratory Animals.

Histological examination

The severity of interstitial fibrosis was evaluated histologically using polarized light microscopy to examine paraffin-embedded sections stained with picrosirius red that identifies interstitial collagen fibers. The tubulointerstitial area stained (expressed as % total area excluding glomeruli and large vessels) was quantified in five random, nonoverlapping cortical fields at 400 magnification using a computer-assisted image analysis system (Optimas image analysis software; Optimas Corp., Bothell, WA, USA).

Interstitial cellularity was characterized and quantified after immunoperoxidase staining for macrophages (F4/80 rat anti-mouse macrophage monoclonal antibody; Serotec Ltd., Oxford, UK), myofibroblasts (peroxidase-conjugated murine anti-human -smooth muscle actin 1A4 monoclonal antibody from Dako Corp., Carpinteria, CA, USA), and proliferating cells (MAS 250 rat anti-BrdU monoclonal antibody from Harlan Sera-Lab Ltd., Loughborough, UK). F4/80 and MAS 250 were detected with peroxidase-conjugated, mouse plasma-absorbed F(ab')₂ goat anti-rat IgG (Accurate Chemical & Scientific Corp., Westbury, NY, USA) using 3,3'-diaminobenzidine (DAB) as the chromogen (Dako Corp.) and methyl green counterstaining. Sections stained with the secondary antibody alone were negative. Staining of the 1A4 antibody was detected using the Enhanced Polymer One-Step Staining (EPOS) reagent (Dako Corp.) as previously described¹⁸.

Total collagen assay

Hydroxyproline concentrations in hydrolysates extracted from accurately weighed frozen kidney samples were chemically measured according to the technique of Kivirikko, Laitinen, and Prockop¹⁹. Total collagen was assumed to contain 12.7% hydroxyproline by weight and final results were expressed as g collagen/mg kidney weight.

Northern blot analysis

Total kidney RNA was isolated using the TRIZOL single-step reagent (GIBCO BRL Life Technologies, Grand Island, NY, USA). RNA samples (20 g) from each individual animal were separated by electrophoresis through 1% agarose gels and transferred to nylon membranes (GeneScreen Plus; New England Nuclear Life Science Products, Boston, MA, USA). The membranes were hybridized in QuickHyb hybridization solution (Stratagene, La Jolla, CA, USA) containing [³²P]dCTP-labeled cDNA probes. After washing, autoradiographs were obtained. The density of each band was quantified using the NIH Image program. The results were adjusted for unequal RNA loading based on the intensity of the ethidium bromide-stained 28s ribosomal bands. Results were expressed in arbitrary densitometric units standardized to a mean value of 1.0 unit for the sham +/+ group mice (or the 3D UUO group when the band density of sham mice was too faint to be measured).

The cDNA probes used were rat PAI-1 from Dr. T. Gelehrter (University of Michigan, Ann Arbor, MI, USA)²⁰, rat uPA from Dr. J. Degen (University of Cincinnati, Cincinnati, OH, USA), mouse tPA from Dr. S. Strickland (State University of New York, Stony Brook, NY, USA)²¹, mouse 2 anti-plasmin from Dr. A. Sappino (University of Geneva, Geneva, Switzerland)²², rat 1(I) procollagen from Dr. S. Thorgeirsson (National Cancer Institute, Bethesda, MD, USA)²³, mouse 1(III) procollagen from Dr. B. deCrombrugghe (M.D. Anderson Cancer Center, Houston, TX, USA)²⁴, and rat TGF-1 from Dr. S. Qian (National Cancer Institute)²⁵.

PAI-1 Western blotting

Kidney samples containing 20 g of protein were separated under reducing condition on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose membranes. Equal loading and transfer of the samples were confirmed by staining the blot with 0.1% amido black in 30% methanol and 10% acetic acid solution. Membranes were stained with sheep anti-mouse PAI-1 IgG (American Diagnostica Inc., Greenwich, CT, USA) followed by peroxidase-conjugated rabbit anti-sheep IgG (Accurate Chemical & Scientific Corp.). Peroxidase activity was detected using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).

Renal plasminogen activator and plasmin activity

Sample preparation

Frozen kidneys pieces (25% of the kidney) were individually ground into a fine powder under liquid nitrogen frozen conditions using a prechilled mortar and pestle. The powder was mixed with homogenizing buffer (50 mmol/L Tris pH 7.6, 1% SDS) and centrifuged (14,000 g) for 10 minutes. The protein concentration of the supernatant was measured using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and aliquots were stored at -80°C.

Plasminogen gel zymography

To detect renal PA activity, kidney supernatants (1 g protein) were separated under nonreducing conditions on 10% SDS-polyacrylamide gels containing plasminogen (10 g/mL) and casein (2 mg/mL) according to the zymographic methods of Roche, Campeau, and Shaw with a few modifications²⁶. After electrophoresis, the gel was washed with 2.5% Triton X-100 and incubated in 0.1 mol/L glycine (pH 8.3) for 15 hours at 37°C. After the gels were stained with Coomassie blue as described by Zehr, Savin, and Hall, PA activity was revealed as clear lytic zones²⁷. The density of each lytic band was quantified using the NIH Image program. Linearization of the assay was ensured using serial dilutions of low molecular weight (33 kD) human uPA (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) to generate a standard curve. In some assays, 1 mmol/L of amiloride was mixed into the Triton solution and glycine buffer in order to distinguish uPA from tPA. Amiloride is known to inhibit uPA but not tPA²⁸. To distinguish the PA from metalloproteinases, the samples were also run on gels without plasminogen.

Chromogenic assay of plasmin activity

Total kidney plasmin activity was measured using a plasmin-specific chromogenic substrate, Chromozym PL (Boehringer Mannheim, Indianapolis, IN, USA), as described by Arnoletti et al²⁹. This substance is specifically cleaved by plasmin into a residual peptide and 4-nitraniline, which can be detected spectrophotometrically. Samples (100 L) containing 80 g of kidney protein, 750 L of 50 mmol/L Tris (pH 8.2), and 100 L of chromogenic substrate were mixed in a cuvette. The absorbance was measured at 405 nm twice over a two-hour interval. The increase in absorbance, corresponding to plasmin activity, was calculated. A standard linear curve was generated with serial dilutions of porcine plasmin (Sigma Chemical Co.). Results were expressed in arbitrary delta spectrophotometric units standardized to a mean value of 1.0 U for sham-operated +/+ mice.

Macrophage chemotaxis assay

Thioglycollate-elicited peritoneal macrophages from five different mice were placed in individual lower wells (35,000 per well) of a microchemotaxis chamber as described by Falk, Goodwin, and Leonard³⁰. Separated from the lower wells by a polycarbonate 5 m pore filter (Millipore, Bedford, MA, USA), chemotactic substances were placed in triplicate in the upper wells. The chamber was incubated at 37°C for four hours in humidified air with 5% CO₂. The filter was removed, methanol fixed, and stained with Diff-Quick (Dade Diagnostics, Aguada, Puerto Rico). Numbers of macrophages in 10 random fields (400) were counted. Added to the upper chamber were formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe, 10^-8 mol/L; Sigma Chemical Co.) as the positive control and media alone (2% albumin HEPES-RPMI) as the negative control and serial dilutions (10^-8 to 10^-13) of mouse recombinant uPA or PAI-1 (American Diagnostica Inc.).

Statistical analysis

Results were expressed as mean 1 SD. Results were analyzed by the Mann Whitney U test. A P value <0.05 was considered statistically significant.

RESULTS

Severity of renal fibrosis

The mean weight of the obstructed left kidneys (corrected for body weight) from the +/+ mice was significantly lower than the -/- mice at 14D, suggesting more severe fibrosis (3.9 0.6 vs. 5.0 0.3 10^-3 mg/mg). Histologically, the degree of interstitial fibrosis, quantified as the percentage of tubulointerstitial area stained by picrosirius red, was significantly less in the PAI-1 -/- mice than in +/+ mice Figure 1. This attenuation in renal fibrosis severity was also evident in the total kidney collagen levels at 7D; sufficient kidney tissue was not available for D14 measurements Figure 2. By linear regression analysis, there was a strong positive correlation between the tubulointerstitial area stained with picrosirius red and the total kidney collagen content (r = 0.80, P = 0.000009).

Figure 1.

Sirius red staining of interstitial collagen fibrils. (A–D) These are from plasminogen-activator inhibitor-1 (PAI-1) wild-type (+/+) mice that were sham-operated (sham), and after 3, 7 and 14 days (3D, 7D, and 14D) of unilateral ureteral obstruction (UUO), respectively. (E–H) These are -/- sham, 3D, 7D, and 14D UUO, respectively (400). (I) Data are mean 1 SD % of the interstitial area stained. Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype.

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Figure 2.

Total kidney collagen in () +/+ and () -/- mice. Results are mean 1 SD. *P < 0.05 compared with sham controls of the same genotype.

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Profile of the intrarenal PA/plasmin cascade

Gene and protein expression

Northern blotting for PAI-1 showed a single band at 3.2 kb in the PAI-1 +/+ mice. The band was barely visible in sham-operated +/+ mice but was strongly induced in response to UUO Figure 3. PAI-1 protein was detected by Western blotting of kidney homogenates from UUO, but not sham mice Figure 4. PAI-1 mRNA and protein were undetectable in the kidneys of PAI-1 -/- mice at all time points.

Figure 3.

(A) Representative Northern blot autoradiograph of renal mRNA levels for plasminogen-activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and 2-antiplasmin (2-AP). The graph shows the results of PAI-1 Northern blotting when RNA from each experimental animal was loaded into separate wells, expressed in arbitrary units as mean 1 SD (B). No PAI-1 mRNA band was detected in the kidneys of the -/- mice. *P < 0.05 compared with sham controls of the same genotype. Similar analyses (data not shown) found a significant 50% increase in uPA only at 7D after UUO and a 300 to 400% increase in tPA at 3D, 7D, and 14D after UUO compared with sham kidneys, and a significant 20 to 60% decrease in 2-AP at all time points. There were no differences in the tPA, uPA, and 2-AP responses between the +/+ and -/- mice.

Full figure and legend (76K)

Figure 4.

PAI-1 Western blotting of kidney supernatants identified a definite PAI-1 protein band (53 kD) only in the +/+ mice at 7D and 14D. Amido black staining shown in the lower portion of this figure confirmed equivalency of protein loading and transfer.

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Renal uPA mRNA was abundantly expressed in the sham mice. Renal tPA mRNA was expressed at low levels in sham kidneys. Renal mRNA levels for both plasminogen activators were increased after UUO surgery mice, but to a similar degree in the +/+ and -/- mice Figure 3.

Since 2 antiplasmin (2-AP) is the major plasmin inhibitor expressed in normal kidneys (personal observations)²², its expression was examined and found to be significantly decreased after UUO surgery Figure 3. The mean message levels at 3D and 7D UUO were higher in the -/- than the +/+ mice, but the difference was not statistically significant (P = 0.12 at 3D, P = 0.07 at 7D).

Enzymatic activity

Plasminogen gel zymography produced a major lytic band at 40 kD and a smaller band at 28 kD Figure 5. This activity was identified as uPA based on its size and by the complete disappearance of both bands when plasminogen was eliminated from the gel or when the gel was treated with amiloride (data not shown). The 40 kD uPA activity increased significantly in UUO mice at 7D and 14D compared with sham mice; there was no difference between +/+ and -/- mice. Similarly, intra-renal plasmin activity increased significantly after UUO surgery. The only difference between +/+ and -/- mice was a paradoxically higher level in the +/+ mice at 3D Figure 6.

Figure 5.

(A) Representative plasminogen gel zymogram. The graph illustrates the mean relative activity after analysis of each experimental animal (B). Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype. Abbreviation is: H.uPA, human urokinase-type plasminogen activator.

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Figure 6.

Renal plasmin activity expressed as mean relative activity 1 SD. Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype.

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Renal expression of profibrotic genes

Northern blotting was performed to evaluate the possibility that genetic manipulations of PAI-1 might alter the expression of other genes currently thought to be involved in renal fibrosis. Renal mRNA levels for TGF-1, 1(I) procollagen, and 1(III) procollagen increased in response to UUO and were significantly higher in +/+ mice than in -/- mice at 3D and 7D UUO Figure 7.

Figure 7.

(A) Representative Northern blot autoradiograph of renal mRNA levels for 1(I) procollagen (Col I) and 1(III) procollagen (Col III) and transforming growth factor- (TGF-). The graphs show the results when RNA from each experimental animal was loaded into separate wells and probed, expressed in arbitrary units as mean 1 SD (B). Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype.

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Interstitial cellularity

The number of interstitial macrophages and interstitial myofibroblasts increased in response to UUO, but the intensity of the response was significantly attenuated in the -/- mice at 3D and 7D compared with the +/+ mice Figures 8 and 9. Similar results were obtained when the number of F4/80-positive interstitial macrophages was manually counted after indirect immunofluorescence staining using ethidium bromide to identify cell nuclei as previously described (data not shown)³¹. Very few BrdU-positive tubulointerstitial cells were observed in sham kidneys. There was a striking 30- to 60-fold increase in the number of BrdU-positive interstitial cells 3, 7, and 14 days after UUO surgery in both genotypes, suggesting that the difference in the number of interstitial macrophages and myofibroblasts between +/+ mice and -/- mice was not due to different rates of in situ proliferation (data not shown). There was a significant positive correlation between interstitial macrophages and renal TGF- mRNA levels (r = 0.71, P = 0.000135) and between renal TGF- mRNA levels and interstitial myofibroblasts (r = 0.65, P = 0.000135).

Figure 8.

F4/80+ interstitial macrophages. (A–D) These are +/+ sham, 3D, 7D, and 14D UUO, respectively. (E–H) These are -/- sham, 3D, 7D, and 14D UUO, respectively (400). (I) The graph is % interstitial area stained expressed as mean 1 SD. *P < 0.05 compared with sham controls of the same genotype.

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Figure 9.

-Smooth muscle actin (-SMA)-positive interstitial myofibroblasts. (A–D) These are +/+ sham, 3D, 7D, and 14D UUO respectively. (E–H) These are -/- sham, 3D, 7D, and 14D UUO, respectively. (I) The graph is % interstitial area stained expressed as mean 1 SD. *P < 0.05 compared with sham controls of the same genotype (400).

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Macrophage chemotaxis for PAI-1

A reproducible macrophage chemotactic response was observed to recombinant mouse PAI-1 at a concentration of 10^-10 to 10^-11 mol/L Figure 10.

Figure 10.

Murine macrophage chemotactic response to murine PAI-1 and uPA. Symbols are: () PAI-1; () uPA; (#) PAI-1 + urokinase receptor macrophages (uPAR -/- M) Results are the mean values of five separate experiments. The (+) control line indicates the mean number of macrophages recruited in response to 10^-8 mol/L f-met-leu-phe (42 7 in 5 experiments). The (-) control line indicates the mean number of macrophages recruited in response to media alone (18 6 in 5 experiments). In three separate experiments using peritoneal M from mice with a genetic deficiency of the urokinase receptor (uPAR-/-), a similar chemotactic response to PAI-1 was observed (# linked with a broken line illustrate mean values). *P < 0.05 compared with the negative control.

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DISCUSSION

This study found that the development of interstitial fibrosis in mice with complete ureteral obstruction was associated with marked induction in renal PAI-1 expression. Furthermore, genetic deficiency of PAI-1 had a protective effect, significantly attenuating the severity of fibrosis at least until 14 days. While the obstructed wild-type kidney is already severely damaged by 14 days, it is unknown whether redundant genetic pathways would have compensated for PAI-1 deficiency after longer periods of obstruction. Genetic manipulations of PAI-1 activity also have been shown to influence the severity of pulmonary fibrosis³².

Additional studies, undertaken to determine the mechanism underlying the protective effect of PAI-1 deficiency, yielded some unanticipated results. We were unable to show that the total renal uPA and plasmin activities were decreased as a consequence of PAI-1 production in the kidneys of the +/+ mice. Normally, uPA is synthesized in large quantities by renal tubules, and it is secreted into the urinary space. In the face of tubular damage caused by obstruction, it is likely that this protease also leaks into the interstitial space. Within the interstitium, uPA may degrade some matrix proteins, but its major antifibrotic actions are thought to be a consequence of its ability to generate plasmin, which activates latent matrix-degrading proteases of the metalloproteinase family. It is clear that the kidney is a rich source of plasminogen activator activity, the level of which actually increased rather than declined in response to ureteral obstruction despite an 8- to 16-fold increase in the expression of its inhibitor PAI-1 in the +/+ mice. This may be due in part to the fact that 2-AP, the most abundant known renal uPA inhibitor, was suppressed following UUO. The assays used in this study measured total kidney uPA and plasmin activity, and it is still possible that there were significant localized differences in the interstitial compartment that were not detected. In addition, it is also conceivable that some of the uPA and plasmin activity detected in these assays was due to the activation of the proenzymes during the isolation procedure. For these reasons, it is not possible to conclude with certainty that the protective mechanisms resulting from PAI-1 deficiency were independent of its inhibitory effects on interstitial PA activity.

The results of this study suggest a novel role for PAI-1 in the recruitment of interstitial macrophages and myofibroblasts, and that this activity is an important reason for the blunted fibrogenic response observed in the PAI-1–deficient mice. In support of this hypothesis is the observation that the delayed appearance of interstitial macrophages and myofibroblasts was associated with lower levels of expression of the genes encoding TGF-1 and interstitial collagens. Both interstitial macrophages and myofibroblasts are a potential source of TGF- during the active phase of renal fibrosis. In the present study, there was a strong positive correlation between both of these populations of interstitial cells and renal TGF- mRNA levels, suggesting that the antifibrotic effects of the PAI-1–deficient state were closely linked with the delayed recruitment of TGF-–producing interstitial cells. Indeed, several other studies have suggested a role for PAI-1 in cell migration, but its actions appear to be complex with both facilitatory and inhibitory effects reported. PAI-1 may bind to vitronectin and block vitronectin-integrin interactions, leading to the inhibition of cellular migration along extracellular matrices^33,34. However, other in vitro studies report that binding of PAI-1 to vitronectin actually interferes with vitronectin binding to the urokinase receptor and facilitates cell migration³⁵.

Additional evidence supporting a role for PAI-1 in the promotion of cell migration comes largely from cancer cell biology. For many human cancers, high tumor expression of PAI-1 is a poor prognostic indicator, predictive of more aggressive local invasion and metastasis³⁶. In a recent study of transplanted malignant keratinocytes, genetic deficiency of PAI-1 prevented local tumor invasion³⁷. This beneficial effect was associated with a dampened angiogenic response. The possibility that PAI-1 modulates vascular and perivascular cellular responses in the kidney deserves further consideration. While the origin of renal interstitial myofibroblasts remains controversial, one possibility is that they are derived from perivascular cells that migrate into the interstitium in response to injury^2,38. The interstitial infiltrate of macrophages is primarily a consequence of the migration of circulating mononuclear cells into the interstitium, a process in which the peritubular capillary endothelium plays a significant role through the production of chemokines and the expression of leukocyte adhesion molecules. While some in situ proliferation of interstitial myofibroblasts and macrophages appears to occur^39,40, the number of proliferating tubulointerstitial cells was not altered by the absence of PAI-1 in the present study. It is possible that further studies will establish a role for PAI-1 in the cascade of events that transform the interstitial cellularity to a fibrosis-promoting phenotype.

Another obvious consideration is the possibility that PAI-1 itself might function as a chemoattractant, even though a cellular receptor for PAI-1 has not yet been described. In this study, we observed a reproducible chemotactic response of activated peritoneal macrophages to recombinant PAI-1 over a narrow concentration range of 10^-11 to 10^-10 mol/L. The observed desensitization of directed motility at higher PAI-1 concentrations is a typical feature of chemotactic molecules⁴¹. Similar results were obtained when peritoneal macrophages were isolated from mice with a genetic deficiency of the uPA receptor, indicating that the chemotactic response to PAI-1 is independent of this receptor. Whether this concentration of PAI-1 is within the physiologic range that might be encountered in diseased kidneys is currently unknown, but these findings suggest another plausible explanation for the delayed interstitial influx of monocytes after UUO in PAI-1–deficient mice. Presently, it is not possible to dismiss the possibility that PAI-1 may modulate interstitial macrophage recruitment indirectly by regulating the generation of other chemotactic factors such as fibrin, monocyte chemoattractant protein-1, complement components C3a and C5a, RANTES, and/or osteopontin, for example. Ongoing studies are investigating the effects of PAI-1 deficiency on the expression of these molecules in damaged kidneys.

In summary, up-regulated expression of PAI-1 in response to renal obstruction appears to play an important role in the ensuing fibrogenic response. The results of the present study suggest that an important profibrotic effect of PAI-1 is its ability to promote the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.

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Acknowledgments

This work was funded by grant support from the National Institutes of Health DK-54500 (A.A.E.). We are grateful to Dr. P. Carmeliet for providing the mice and to the following individuals for technical advice: Dr. R.D. Kenagy with gel zymography, Dr. M. Haraguchi with the plasmin assay, and Dr. J. Hughes with UUO surgery. Part of this work was previously published in abstract form (J Am Soc Nephrol 11:535A, 2000).