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J Am Soc Nephrol 14:1254-1271, 2003
© 2003 American Society of Nephrology

Urokinase Receptor Deficiency Accelerates Renal Fibrosis in Obstructive Nephropathy

Guoqiang Zhang*, Heungsoo Kim*, Xiaohe Cai*, Jesús M. López-Guisa*, Charles E. Alpers{dagger}, Youhua Liu{dagger}, Peter Carmeliet§ and Allison A. Eddy*

*University of Washington and Children’s Hospital and Regional Medical Center, Division of Nephrology, Seattle, Washington; {dagger}Department of Pathology, University of Washington, Seattle, Washington; {ddagger}Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania; and §The Center For Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.

Correspondance to Dr. Allison A. Eddy, Children’s Hospital and Regional Medical Center, Division of Nephrology, Mail Stop 5G-1, 4800 Sand Point Way NE, Seattle, WA 98105. Phone: 206-987-2524; Fax: 206-987-2636;


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The urokinase cellular receptor (uPAR) recognizes the N-terminal growth factor domain of urokinase-type plasminogen activator (uPA) and is expressed by several cell types. The present study was designed to test the hypothesis that uPAR regulates the renal fibrogenic response to chronic injury. Groups of uPAR wild-type (+/+) and deficient (-/-) mice were investigated between 3 and 14 d after unilateral ureteral obstruction (UUO) or sham surgery. Not detected in normal kidneys, uPAR mRNA was expressed in response to UUO in the +/+ mice. By in situ hybridization, uPAR mRNA transcripts were detected in renal tubules and interstitial cells of the obstructed uPAR+/+ kidneys. The severity of renal fibrosis, based on the measurement of total collagen (13.5 ± 1.5 versus 9.8 ± 1.0 µg/mg kidney on day 14; -/- versus +/+) and interstitial area stained by Masson trichrome (22 ± 4% versus 14 ± 3% on day 14; -/- versus +/+) was significantly greater in the uPAR-/- mice. In the absence of uPAR, renal uPA activity was significantly decreased compared with the wild-type animals after UUO (62 ± 20 versus 135 ± 13 units at day 3 UUO; 74 ± 17 versus 141 ± 16 at day 7 UUO; 98 ± 20 versus 165 ± 10 at day 14 UUO; -/- versus +/+). In contrast, renal expression of several genes that regulate plasmin activity were similar in both genotypes, including uPA, tPA, PAI-1, protease nexin-1, and {alpha}2-antiplasmin. Worse renal fibrosis in the uPAR-/- mice appears to be TGF-{beta}-independent, as TGF-{beta} activity was actually reduced by 65% in the -/- mice despite similar renal TGF-{beta}1 mRNA levels. Significantly lower levels of the major 2.3-kb transcript and the 69-kd active protein of hepatocyte growth factor (HGF), a known anti-fibrotic growth factor, in the uPAR-/- mice suggests a potential link between HGF and the renoprotective effects of uPAR. These data suggest that renal uPAR attenuates the fibrogenic response to renal injury, an outcome that is mediated in part by urokinase-dependent but plasminogen-independent functions. E-mail: allison.eddy@seattlechildrens.org


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Progressive renal disease, characterized histologically by tubular atrophy and the accumulation of extracellular matrix proteins in the renal interstitium, is associated with declining renal function (1,2). In addition to increased matrix protein synthesis, impaired degradation of interstitial matrix proteins also appears to play an important role in renal fibrogenesis (3). Although the mechanisms regulating matrix turnover within the renal interstitium are poorly understood, recent studies suggest that the plasmin cascade plays a significant role (4). In addition to their well-known fibrinolytic activity, the plasminogen activator/plasmin enzymes also have direct and indirect extracellular matrix-degrading actions (5,6). Somewhat paradoxically, plasmin also activates the latent fibrogenic cytokine TGF-{beta} in vitro, but whether this is a significant in vivo action of plasmin is unclear (7). The rate-limiting step for plasmin generation is the activity of tissue-type (tPA) and/or urokinase-type (uPA) plasminogen activators. PA activity is tightly regulated at several levels, including pro-enzyme synthesis, extracellular activation of the latent enzymes, and their inhibition by specific inhibitors such as plasminogen activator inhibitor-1 (PAI-1), PAI-2, and possibly protease nexin-1. Within normal kidneys, high levels of uPA are synthesized by renal tubules. Generally undetected in normal kidneys, PAI-1 is expressed de novo during the active phase of renal fibrosis (reviewed in reference 8). Although mice with the PAI-1 null mutation develop less severe renal (9) and pulmonary fibrosis (10) than do wild-type mice, it remains unclear whether the blunted fibrogenic response is entirely due to inhibition of plasminogen activator/plasmin proteolytic activity or also related to the pro-inflammatory and pro-angiogenic effects of PAI-1.

A cellular receptor has been identified for the N-terminal growth factor domain of uPA (uPAR), also known as CD87 (11,12). It is expressed by cells of several lineages, including lymphohematopoietic cells (monocytes, neutrophils, and activated T cells), resident kidney cells (glomerular and tubular epithelial cells and mesangial cells), endothelial cells, fibroblasts, and myofibroblasts (5,13–18). This highly glycosylated 50-kD to 65-kD protein is linked to the plasma membrane by glycosylphosphatidylinositol (GPI). The uPAR binds to both the latent and active form of uPA. Once receptor-bound, the latent enzyme can be activated while the active enzyme retains its enzymatic activity. The inhibitor PAI-1 may also bind to the receptor-bound enzyme, an interaction that promotes internalization and degradation of uPA and PAI-1 (5,19). Soluble forms of uPAR also exist, generated by proteolytic cleavage of the transmembrane domain (20).

It is now evident that uPAR is a multifunctional receptor that is involved not only in cell-surface uPA activity and plasmin generation, but also in mediating protease-independent effects, including cell adhesion and migration and outside-in signaling (12,21). Less clear is the role of receptor-bound uPA and subsequent plasmin generation in the context of tissue remodeling. In particular, little is known about the expression and function of uPAR during renal fibrosis. Immunohistochemical studies have reported uPAR expression on normal human tubules in one study (18) but not in another (16). Increased kidney uPAR expression has been described in several renal disease states such as endotoxemia (15,22), acute tubular necrosis (16), thrombotic microangiopathy (16), nephrotoxic serum nephritis (23), pyelonephritis (22), and chronic allograft rejection (24). The present study was designed to investigate the functional role of uPAR in the renal fibrogenic response to sustained injury by investigating the response to ureteral obstruction in uPAR-deficient mice compared with wild-type mice of the same genetic background.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Experimental Design
UPAR-/- and +/+ mice on a C57Bl6 background were bred in our animal facility and allowed to grow to a minimum weight of 20 g before the initiation of the study (25). The genotypes of the mice were confirmed by Southern blot analysis of DNA extracted from tails. Groups of weight-matched and gender-matched uPAR-deficient and wild-type mice were studied 3, 7, and 14 d after unilateral ureteral obstruction (UUO) and 7 d after sham surgery (n = 8 per group). UUO surgery was performed under general anesthesia. The left ureter was ligated with 4.0 silk at two separate points in the UUO groups. All mice were sacrificed by exsanguination under general anesthesia. All procedures were performed in compliance with the guidelines established by National Research Council Guide for the Care and Use of Laboratory Animals.

Kidney Tissue Preparation
After exsanguination, the left kidney was procured, the capsule removed, and the kidney weighed. The kidney was divided longitudinally and subdivided. One half was prepared for histologic studies: one piece (approximately one third) was fixed in 10% buffered formalin and paraffin-embedded; the remaining piece (approximately two thirds) was imbedded in Tissue-Tek OCT compound (Sakura Finetek, Torrence, CA) and snap-frozen. Sections from the second half kidney were frozen at -80°C for total collagen measurement (approximately one fourth) and for the total RNA and protein extraction (approximately three fourths).

Analysis of Tubulointerstitial Fibrosis
Total renal collagen was measured biochemically as described previously (26). In brief, an accurately weighed portion of the kidney was homogenized in distilled water, hydrolyzed in 10N HCl and incubated at 110°C for 18 h. The hydrolysate was dried by speed vacuum centrifugation and redissolved in buffer (25 g of citric acid, 6 ml of glacial acetic acid, 60 g of sodium acetate, and sodium hydroxide [17 g in 500 ml], pH 6.0). Total hydroxyproline in the hydrolysate was determined according to the chemical method of Kivirikko et al. (27). Total collagen in the tissue was calculated on the assumption that collagen contains 12.7% hydroxyproline by weight. Final results were expressed as µg/mg kidney wet weight.

Kidney sections (day 14 UUO and sham) were stained with Masson trichrome (Sigma, St. Louis, MO), and the percent aniline blue-stained tubulointerstitial area was measured using a point-counting method (28).

Immunohistology
Immunohistochemical studies were performed on 4-µm paraffin-embedded renal sections. Primary antibodies used were rabbit anti-human LDL receptor-related protein (LRP) provided by Dr. D.K. Strickland, American Red Cross, Rockville, MD (29), rabbit anti-mouse urokinase (American Diagnostica Inc., Greenwich, CT), rabbit anti-human TGF-{beta} (Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-human HGF (30). Immunoperoxidase staining was performed using the ABC ELITE kit (Vector Laboratories Inc., Burlingame, CA). Sections stained with the secondary antibodies alone were negative. For HGF staining, the primary and secondary antibodies were pre-complexed before incubation with the tissue sections to minimize crossreactivity (31).

To evaluate the extent of tubular injury, sections were stained with biotinylated Phaseolus vulgaris agglutinin-E (PHA-E) (Vector Laboratories Inc.), a lectin that binds to proximal tubular brush border (32) or with a mouse anti-kidney-specific cadherin (Ksp) monoclonal antibody (Zymed Laboratories Inc, San Francisco, CA), which is expressed on the basolateral membrane of collecting ducts (33). PHA-E staining was used to grade tubular injury on a scale of 1 to 4 as described by Mizuno et al. (34). Loss of Ksp-cadherin expression was determined as a measure of early tubular injury and expressed as % positive tubules.

In Situ Hybridization
uPAR in situ hybridization was performed as described previously using a 35S-labeled riboprobe prepared using the Riboprobe Combination System-T3/T7 RNA Polymerase kit (Promega Corp., Madison, WI) (35). In brief, formalin-fixed, paraffin-embedded 4-µm kidney tissue sections were deparaffinized and rehydrated using standard procedures. Sections were washed with 0.5x SCC and digested with 10 µg/ml proteinase K (Sigma). Prehybridization was performed for 2 h by adding 50 µl of prehybridization buffer (0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM ethylenediaminetetraacetic acid, 1x Denhardt solution, 10% dextran sulfate, 10 mM dithiothreitol, and 50 µg/ml yeast tRNA). Hybridization was initiated by adding 500,000 cpm of the 35S-labeled uPAR riboprobe (sense or anti-sense) in 50 µl of prehybridization buffer and incubated overnight at 50°C. Sections were treated with RNase A (20 µg/ml; Sigma) followed by three high-stringency washes in 0.1x SSC/0.5% Tween 20 (Sigma) for 40 min at 50°C and several 2x SSC washes. After the tissue was dehydrated and air-dried, it was dipped in NTB2 emulsion, (Kodak, Rochester, NY) and developed in the dark for 10 wk. After development, the sections were counterstained with hematoxylin and eosin, dehydrated, and cover-slipped.

Northern Blot Analysis
Total kidney RNA was isolated by the phenol/guanidine isothiocyanate extraction method using TRIzol-BRL reagent (Life Technologies). Total kidney RNA (18 µg) from each experimental animal was loaded into individual wells and separated by 1.0% agarose formaldehyde gel electrophoresis. A photomicrograph of the ethidium bromide-stained gel was obtained to determine RNA loading equality. The RNA was transferred to a hybridization membrane (GeneScreen Plus; New England Nuclear Life Science Products, Boston, MA) and ultraviolet light crosslinked (UV Crosslinker; Hoeffer Scientific Instruments, San Francisco, CA). Complementary DNA probes were radiolabeled with 32P dCTP (3000 Ci/mmol) by random priming with T7 Quick Prime kit (Pharmacia Biotech, Piscataway, NJ). The blots were hybridized with the radiolabeled cDNA probes using the QuickHyb hybridization buffer (Stratagene, La Jolla, CA). Autoradiographs were obtained and the density of each band quantified using the NIH Image program. The 18-s ribosomal bands in the ethidium bromide-stained gels were used to adjust for RNA loading equality as described previously (36).

The cDNA probes used were murine uPAR1 (from Dr. Niels Behrendt, Finsen Laboratory, Copenhagen, Denmark) (37), rat fibronectin lambda-rlf-1 (from Dr. R. Hynes, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA) (38), mouse {alpha}1(I) procollagen (from Dr. S. Thorgeirsson, National Cancer Institute, Bethesda, MD) (39), rat TGF-{beta}1 (from Dr. S.W. Qian, National Cancer Institute) (40), mouse hepatocyte growth factor (41), rat PAI-1 (from Dr. T.D. Gelehrter, University of Michigan, Ann Arbor, MI) (42), rat uPA (from Dr. J. Degen, Children’s Hospital Research Foundation, University of Cincinnati, Cincinnati, OH), mouse tPA (from Dr. D.S. Strickland, The Rockefeller University, New York, NY) (43), mouse plasminogen (American Tissue Culture Collection, Rockville, MD), mouse protease nexin-1 (from Dr. Vejsada, University of Geneva, Geneva, Switzerland) (44), and mouse {alpha}2-antiplasmin (from Dr. A. Sappino, University of Geneva, Geneva, Switzerland) (45).

Protease Activity
Protein was isolated from kidney tissue that had been stored at -80°C. Pieces were individually ground into a fine powder under liquid nitrogen conditions, using a mortar and pestle that had been prechilled with dry ice. For gelatin zymography, the powder was homogenized in extraction buffer (0.05 M Tris, 0.01 M CaCl2, 2.0 M guanidine HCl, 0.2% Triton X-100, pH 7.5) and dialyzed using dialysis membrane Spectra/PorR 1 (Spectrum Medical Industries, Inc., Houston, TX) against 0.05 M Tris, 0.2% Triton X-100, pH 7.5, for 48 h at 4°C. For casein plasminogen zymography, the powder was mixed with homogenizing buffer (50 mmol/L Tris, pH 7.6, 1% SDS). Individual samples were then centrifuged for 5 min (14,000 x g), and the protein concentration was measured in the supernatants using the Bradford protein assay kit (Bio-Rad, Hercules, CA). Samples were aliquoted and stored at -80°C for zymographic studies.

MMP-9 and MMP-2 activity were measured by gelatin zymography according to the method reported by Kenagy et al. (46,47). In brief, protein samples (10 µg/well) were loaded without heating onto a 7% polyacrylamide gel containing 1 mg/ml porcine skin gelatin (Sigma) as substrate. Molecular markers and human MMP-2 and MMP-9 standards (Chemicon International Inc., Temecula, CA) were also loaded into the outer wells. After protein separation by electrophoresis, the gel was rinsed in 2.5% Triton X-100 at room temperature with gentle shaking for 30 min. After incubation for 17 to 20 h at 37°C in a solution containing 50 mM Tris and 10 mM CaCl2, pH 7.8, the gel was stained with 0.002% Coomassie blue and photographed. The size of each lytic band was measured using the NIH image analysis program.

Casein plasminogen zymography was performed to evaluate renal uPA and tPA activity using the methods of Roche et al. (48) with minor modifications as described previously (9). The procedure used was similar to gelatin zymography, except that the zymography gel was made of 10% SDS-polyacrylamide containing 2 mg/ml {alpha}-casein and 10 µg/ml plasminogen (Sigma). Molecular weight markers and human urokinase standards (Calbiochem Co., San Diego, CA) were loaded into the outer wells. PA-specific bands were verified by their disappearance when re-run in an identical gel that lacked plasminogen.

Total kidney plasmin activity was measured using a plasmin-specific chromogenic substrate, Chromozym PL (Boehringer Mannheim, Indianapolis, IN) as described previously (9).

TGF-{beta} Bioactivity
TGF-{beta} bioactivity was measured in kidney protein extracts using a TGF-{beta}–responsive mink lung epithelial cell (MLEC) line (a generous gift from Dr. Daniel B. Rifkin, New York University Medical Center, New York, NY) (49). This cell line was generated by fusing a truncated TGF-{beta}-inducible PAI-1 promoter to a firefly luciferase reporter gene and transfected into MLEC. This bioassay is specific for active TGF-{beta} with a detection limit of approximately 5 to 10 pg/ml. Kidney protein samples (15 µg) diluted in serum-free media (triplicate samples from three individual animals per group) were added directly to monolayers of confluent MLEC cultures. A standard curve of TGF-{beta}1 activity was generated using serial dilutions of recombinant human active TGF-{beta}1 (ED50 = 0.05 to 0.1 ng/ml; Amersham Pharmacia). After overnight incubation at 37°C in 5% CO2, cells were harvested and luciferase activity measured using the Enhanced Luciferase Assay Kit (Pharmingen, San Diego, CA).

HGF Western Blotting
Pro-HGF and the active form of the HGF protein ({alpha} chain) were detected by Western blot analysis as described by Grenier et al. (50). Protein samples (40 µg) were separated by 10% SDS-PAGE under reducing conditions. The proteins were transferred to a nitrocellulose membrane, and the immunoreactive protein was visualized using ECL enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The primary antibody was monoclonal anti-human HGF (clone 8), known to crossreact with rat HGF (30); the secondary antibody was HRP-conjugated goat anti-mouse IgG (Sigma). Protein loading equality was determined by amido black staining.

Statistical Analyses
All 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
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 Materials and Methods
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Discussion
 References
 
Renal Expression of uPAR and LDL-Receptor Related Protein
Renal uPAR mRNA was undetectable by Northern blotting in sham-operated kidneys and remained undetectable in all UUO kidneys from uPAR-/- mice. UPAR mRNA was expressed in UUO kidneys from the uPAR+/+ mice animals after 3 d of obstruction (Figure 1). In situ hybridization studies on kidneys after 7 and 14 d of obstruction identified uPAR transcripts in uPAR+/+ kidneys, especially in tubular and interstitial cells (Figure 2). Expression of LRP by interstitial cells, an endocytosis receptor that functions as an uPAR co-receptor, was increased to a greater extent in the uPAR+/+ mice than the uPAR-/- mice after ureteral obstruction (Figure 3).



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  		Figure 1. Renal urokinase cellular receptor (uPAR) gene expression. Kidney total RNA from individual mice (18 µg each) was separated and transferred to a nylon blot. The membrane was probed with 32P-dCTP–labeled mouse uPAR1 cDNA and exposed for 120 h. A 1.5-kb uPAR mRNA band was detected in kidneys of uPAR+/+ mice after unilateral ureteral obstruction (UUO).

 


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  		Figure 2. uPAR in situ hybridization. Using 35S-labeled uPAR anti-sense and sense probes, the site of uPAR gene expression was determined after UUO surgery. Anti-sense probes failed to produce positive signals with sham uPAR+/+ (A) and uPAR-/- (B) kidneys and uPAR-/- obstructed kidneys (D). After 7 d of UUO, several tubular cells (examples indicated with arrows) and interstitial cells (highlighted with figure insert) were positive, as indicated by the black silver grains (C). Low levels of background activity are indicated by hybridization of obstructed uPAR+/+ (E) and uPAR-/- (F) kidneys with uPAR sense probes. Magnification: x400.

 


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  		Figure 3. Renal expression of LDL receptor-related protein (LRP) by interstitial cells detected by immunohistochemical staining. In comparison with sham-operated uPAR+/+ (A) and uPAR-/- (B) kidneys, the number of LRP-positive interstitial cells increased after 7 d of ureteral obstruction, although to a greater extent in the uPAR+/+ kidneys (C) compared with the uPAR-/- kidneys (D). Magnification: x400.

 
Severity of Tubulointerstitial Fibrosis
In response to UUO, total kidney collagen increased with time in the mice of both genotypes, reaching a 2.6-fold to 3.6-fold increase relative to the sham kidneys by day 14 UUO (uPAR+/+ and -/-, respectively; Figure 4). The increase in kidney collagen was significantly higher in the uPAR-/- mice: 7.6 ± 0.9 compared with 6.3 ± 0.6 µg/mg kidney at day 7 UUO and 13.5 ± 1.5 versus 9.8 ± 1.0 µg/mg kidney at day 14 UUO (uPAR-/- versus uPAR+/+; P < 0.05). Evaluation of the interstitial area stained blue with Masson trichrome also demonstrated that interstitial fibrosis was significantly more extensive in the uPAR-/- kidneys (Figure 4). Tubular injury was more severe in the uPAR-/- mice. The number of Ksp-cadherin–positive collecting ducts was significantly reduced in the uPAR-/- group within 3 d after UUO (Figure 5), and they were undetectable in both genotypes by 14 d. Using PHA-E lectin staining to evaluate late proximal tubular damage, even after 14 d of obstruction the uPAR+/+ mice had less extensive tubular destruction (Figure 5).



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  		Figure 4. Renal fibrosis severity. The mean total kidney collagen content expressed as µg/mg wet kidney weight was significantly higher in uPAR-/- mice (dotted line) than in uPAR+/+ mice (solid line) by day 7 and day 14 of UUO (A). The interstitial area stained blue by Masson trichrome (day 14) was significantly greater in the uPAR-/- mice (B). Light photomicrographs of Masson trichrome-stained uPAR+/+ (C) and uPAR-/- (D) sham kidneys and uPAR+/+ (E) and uPAR-/- (F) obstructed kidneys illustrate the difference in the extent of interstitial fibrosis between the two genotypes in response to ureteral obstruction. Magnification: x400. Values in the graphs (A and B) represent the mean ± 1 SD (n = 6 per group; *P < 0.05, uPAR-/- versus uPAR+/+ mice).

 


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  		Figure 5. Tubular damage severity. Immunohistochemical staining with the biotinylated lectin Phaseolus vulgaris agglutinin-E (PHA-E) was used to grade the severity of proximal tubular damage (A through F). Binding of PHA-E was limited to the proximal tubular brush border of normal sham kidneys (C and F; uPAR+/+). After 14 d of ureteral obstruction, proximal tubular damage was evident as characterized by brush border loss, epithelial cell simplification, dilation of tubular lumina often with cast formation, and tubular atrophy (A, B, D, and E). Compared with uPAR+/+ mice (A and D), proximal tubular damage was more extensive in the uPAR-/- mice (B and E). The results are shown graphically, expressed as the mean score ± 1 SD; * P < 0.05 (M). The Ksp-cadherin is detected on collecting ducts of normal sham kidneys by immunohistochemical staining (G and J; uPAR+/+ and uPAR-/-, respectively). Three days after UUO, collecting ducts were ectatic and epithelial injury was evident by loss of Ksp-cadherin expression (H and K; uPAR+/+ and uPAR-/-, respectively). Expressed as percent positive tubules ± 1 SD, the loss of Ksp-cadherin was more extensive in the uPAR-/- mice (open bars) compared with the UPAR+/+ mice (solid bars). After 7 d of UUO, very few Ksp-1–positive tubule were detected in mice of both genotypes (I and L; uPAR+/+ and uPAR-/-, respectively); * P < 0.05, UUO versus sham of the same genotype; + P < 0.05, uPAR-/- versus uPAR+/+ mice (N). Magnification: x100 in A through C; x400 in D through L.

 
Renal Expression of PA and PA Inhibitor Genes
Renal expression of plasminogen activator genes was increased to a similar extent in the mice of both genotypes in response to UUO (twofold and threefold increase in uPA and tPA, respectively, at day 7 UUO; Figures 6 and 7). Renal mRNA levels of the two renal serine protease inhibitors, PAI-1 and protease nexin-1, were also strongly induced in response to UUO and to a similar level in uPAR+/+ and -/- mice (Figures 6 and 7). In contrast, renal mRNA levels for the plasmin inhibitor {alpha}2-antiplasmin were significantly downregulated in mice of both genotypes in response to UUO (Figures 6 and 7). Renal plasminogen mRNA was undetectable in all kidneys.



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  		Figure 6. Renal expression of PA and PA inhibitor genes. Representative Northern blot from day 7 studies illustrates specific bands for plasminogen activators uPA (A) and tPA (B), and the inhibitors PAI-1 (C), protease nexin-1 (D), {alpha}2-antiplasmin (E). The changes in response to UUO were similar between the uPAR+/+ and -/- genotypes except for {alpha}2-antiplasmin levels that were downregulated to a lesser extent in the uPAR-/- mice (P < 0.05). The bands of 18S rRNA in the ethidium bromide stained gel showed the equality of RNA loading.

 


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  		Figure 7. Renal expression of PA and PA inhibitor genes. Bar graphs show the results of the quantitative densitometric analysis of the Northern blot studies, as illustrated in Figure 5 for day 7. Shaded bars represent the uPAR+/+ groups; open bars are the uPAR-/- groups. Results are means ± 1 SD expressed in arbitrary densitometric units, with one unit representing the mean value obtained for the sham uPAR+/+ kidneys, except for PAI-1, where gene expression was not detected in the sham kidneys. * P < 0.05, UUO versus sham of the same genotype; + P < 0.05, uPAR-/- versus uPAR+/+ mice.

 
Renal Protease Activity
As illustrated in casein plasminogen gel zymogram (Figure 8), uPA is the predominant plasminogen activator detected in the kidney after UUO. The activity of both uPA and tPA increased with time after UUO, but both were significantly lower in the uPAR-/- mice compared with uPAR+/+ mice. By immunostaining, uPA protein was detected in renal tubules in a similar pattern in both genotypes, including enhanced expression in dilated atrophic tubules found within fibrotic loci (Figure 8). Renal plasmin activity did not differ between uPAR+/+ and -/- mice: sham: 2.7 ± 0.8 versus 2.4 ± 0.5; day 3 UUO: 3.2 ± 2.4 versus 5.3 ± 1.0; day 14: 2.0 ± 1.5 versus 3.1 ± 2.1 x 10-4 units/ml, respectively.



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  		Figure 8. Zymogram of renal plasminogen activator activity. Lytic bands representing renal tPA (65 kD) and uPA (40 kD) activity, as measured by casein plasminogen zymography in uPAR+/+ and -/- mice (A). Semiquantitative evaluation of the uPA and tPA (B) was performed using the NIH Image program, and the results are expressed as the mean ± 1 SD (n = 3 per group). * P < 0.05, uPAR-/- versus uPAR+/+ mice. Photomicrograph of uPA immunoperoxidase staining, illustrating numerous positive tubules in an uPAR-/- kidney 7 days after UUO. Magnification: x400.

 
Gelatin gel zymography demonstrated that MMP-9 was the most abundant gelatinase in the kidney. After UUO surgery, MMP-9 activity decreased compared with the sham kidneys; MMP-9 activity was significantly lower in the uPAR-/- mice compared with uPAR+/+ mice on days 3 and 14 (Figure 9). In contrast, renal MMP-2 activity increased in response to UUO without a consistent difference between the two genotypes: MMP-2 levels slightly lower (nonsignificant) on days 3 and 7 but higher (significant) by day 14 in the uPAR-/- mice (Figure 9).



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  		Figure 9. Zymogram of renal gelatinase activity. Lytic bands represent renal MMP-9 and MMP-2 activity, as measured by gelatin zymography in uPAR+/+ and -/- mice (A). Semiquantitative measurement of the size of the lytic bands was performed using the NIH Image program and expressed as arbitrary units relative to the lytic activity in the sham kidneys of the uPAR+/+ mice. The results are expressed as the mean standardized activity units ± 1 SD (n = 3 per group). Shaded bars are uPAR+/+ mice; open bars are uPAR-/- mice. * P < 0.05, uPAR+/+ versus uPAR-/- mice.

 
TGF-{beta}1
Renal TGF-{beta}1 mRNA was strongly induced in UUO kidneys from mice of both genotypes after 3, 7, 14 d of obstruction: eightfold, fivefold, and fourfold in the uPAR+/+ mice and sixfold, sevenfold, and fivefold in the uPAR-/- mice at 3, 7, and 14 d, respectively (nonsignificant differences between the genotypes; Figure 10). Immunohistochemical staining of the obstructed kidneys identified TGF-{beta}1 protein primarily in renal tubules and occasional interstitial cells (Figure 10). In contrast to the mRNA levels, TGF-{beta}1 bioactivity was actually significantly lower in the uPAR-/- kidneys compared with the uPAR+/+ kidneys after 7 d of obstruction (Figure 10).



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  		Figure 10. Renal TGF-{beta}1 gene expression and bioactivity. The Northern blot autoradiograph illustrates renal TGF-{beta} mRNA levels 3, 7, and 14 d after UUO surgery (A through C). The lower panel below each Northern blot illustrates the ethidium bromide–stained 18S ribosomal bands that were used to adjust for any RNA loading inequality. By densitometric analysis, the mean values expressed in arbitrary units (uPAR+/+ versus uPAR-/-) were: day 3 UUO: 7.5 ± 1.0 versus 8.5 ± 1.4; day 7 UUO: 5.2 ± 0.5 versus 5.1 ± 0.4; and day 14 UUO: 3.7 ± 0.2 versus 3.6 ± 0.2 (P is nonsignificant at all time points). Renal TGF-{beta} bioactivity, measured after 7 d of obstruction using the MLEC cell line transfected with PAI-1 promoter/luciferase gene, showed significantly greater TGF-{beta} bioactivity in the uPAR+/+ mice (shaded bars) than in the uPAR-/- mice (open bars) (D). The TGF-{beta} bioactivity is expressed as mean ± 1 SD pg/ml of triplicate samples from three mice per group. By immunohistochemical staining, TGF-{beta} protein was present in several renal tubules and occasional interstitial cells as illustrated in a uPAR-/- kidney 7 d after UUO (E). Magnification: x250. + P < 0.05 compared with sham group of the same genotype; * P < 0.05, uPAR-/- versus uPAR+/+ mice.

 
Hepatocyte Growth Factor
In vitro studies have reported that urokinase activates the anti-fibrotic growth factor HGF (51). Seven days after UUO surgery, all four HGF transcripts (6.0, 3.1, 2.3, and 1.5 kb) were significantly increased; levels of the major 2.3-kb transcript were significantly lower in the uPAR-/- mice (Figure 11). By immunohistochemistry, the HGF protein was detected within the interstitium. Western blot analysis (day 7) showed significantly lower levels of active HGF ({alpha} chain) in the uPAR-/- mice despite similar amounts of pro-HGF protein in both genotypes (Figure 11). By day 14, HGF protein could not be detected by Western blotting.



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  		Figure 11. Renal hepatocyte growth factor (HGF) expression. Northern blot autoradiograph illustrates the expression of the four HGF mRNA transcripts 7 d after UUO surgery (A). Quantitative densitometric analysis (corrected for RNA loading using the 18S ethidium bromide-stained bands) found significantly higher levels of the major 2.3-kb band in the uPAR+/+ kidneys (shaded bars) compared with the uPAR-/- kidneys (open bars) (B). Immunohistochemical staining of an uPAR-/- kidney 7 d after UUO demonstrating HGF-positive interstitial cells (C). Magnification: x400. Western blot illustrating kidney levels of pro-HGF and mature HGF {alpha}-chain 3 and 7 d after UUO surgery (D). Quantitative analysis demonstrated lower levels of active HGF in the uPAR-/- mice (open bars) compared with the uPAR+/+ mice (shaded bars) at both time points (E). *P < 0.05, uPAR-/- versus uPAR+/+ mice.

 
Extracellular Matrix Genes
Renal procollagen {alpha}1(I) and fibronectin mRNA level was significantly elevated in mice of both genotypes after UUO compared with sham-operated groups (Figure 12). As shown, no differences were noted between the uPAR-/- and uPAR+/+ mice on day 14 after UUO. Although not shown, matrix gene expression was also similar on days 3 and 7.



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  		Figure 12. Renal matrix gene expression. Fibronectin and procollagen {alpha}1(I) mRNA levels measured by Northern blotting 14 d after UUO surgery and expressed as mean densitometric score ± 1 SD for uPAR+/+ (shaded bars) and uPAR-/- (open bars) kidneys. Differences were not statistically significant between the genotypes. Although not shown, changes in matrix gene expression were also similar in uPAR+/+ and uPAR-/- kidneys 3 and 7 d after UUO. * P < 0.05 compared with sham group of the same genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The uPAR is a multifunctional cellular receptor that is involved in such diverse biologic processes as angiogenesis, inflammation, wound repair, and tumor metastasis (12,21). The results of the present study identify an important modulating role for uPAR during the renal fibrogenic response that develops in response to chronic injury caused by ureteral obstruction. Although there are now several reports of increased uPAR expression in damaged kidneys, the functional significance of these changes remains unclear. In the present study, renal uPAR was significantly increased after ureteral obstruction in wild-type mice, while the absence of uPAR in the uPAR null mice was associated with a more aggressive fibrogenic response. The ability of uPAR to attenuate renal fibrosis appears to be related, at least in part, to enhanced activity of proteases that degrade extracellular matrix proteins, including uPA, tPA, and possibly MMP-9.

The kidney is the major site of production of the uPAR ligand, uPA. Other known ligands are kininogen and vitronectin (12,52). In response to UUO, renal pro-urokinase mRNA levels were significantly increased in mice of both genotypes but uPA activity was significantly higher in the uPAR+/+ animals, consistent with the known ability of uPAR to stabilize uPA activity at cellular surfaces. uPAR binds both the single chain pro-urokinase and the two-chain active enzyme. Even after receptor binding, pro-urokinase can be activated while the active enzyme is stabilized. The specific mechanism(s) of pro-urokinase activation within the kidney is unknown; several enzymes can achieve this function in vitro, including plasmin, kallikrein, cathepsins, matrix metalloproteinase-3, and Pump-1 metalloproteinase (53). In the present study, the renal expression of uPAR in the wild-type mice after UUO was associated with significantly higher levels of uPA activity than was observed in the kidneys lacking uPAR.

Urokinase initiates several extracellular events that could accelerate the rate of degradation of matrix proteins. It has limited ability to directly degrade some matrix proteins such as fibronectin (54). It also activates certain latent matrix metalloproteinases, including membrane type (MT)-MMP-1 and MT-MMP-2 (55). However, it is the ability of uPA to catalyze the conversion of plasminogen to plasmin and the subsequent plasmin-dependent activation of metalloproteinases, especially interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), that has been considered to be the primary pathway whereby uPA promotes the degradation of extracellular matrix (56). Significantly less MMP-9 activity, the predominant renal gelatinase, was detected in the uPAR-/- kidneys 3 and 14 d after UUO, which may be one factor contributing to the greater extent of renal fibrosis. Whether alterations in MMP-1 or MMP-3 activity are involved remains speculative, as we have not been able to detect MMP-1 or MMP-3 activity using kidney protein extracts and casein and gelatin zymography.

Recent fibrosis studies in plasminogen-deficient mice have yielded conflicting data and suggest that the plasmin-dependent effects could be tissue-specific. Accelerated fibrosis has been reported in the model of bleomycin-induced pulmonary fibrosis in mice genetically deficient in plasminogen (57), while renal fibrosis induced by UUO was not accentuated in the absence of plasminogen (58). By contrast, in acute glomerular injury associated with fibrin deposition, plasminogen deficiency exacerbates the disease severity (59). In the present study, plasminogen mRNA was not detected in any of the kidneys, and measured renal plasmin activity was unaltered by genotype. We cannot discount the possibility that technical limitations of the plasmin assay accounted for the failure to detect genotype-dependent differences in renal plasmin activity. Alternatively, other regulators of plasmin activity may overshadow plasminogen activator activity in this model. Paradoxically, plasmin may also activate latent TGF-{beta}, at least in vitro (7); whether this is a significant in vivo response is unclear. In the present study, TGF-{beta} bioactivity was actually higher in the wild-type mice, yet the net effect of upregulated uPAR expression was enhanced PA activity and less interstitial collagen deposition.

Urokinase has been reported to induce the expression and activation of HGF, a growth factor with important anti-fibrotic activities (51). HGF has been reported to attenuate renal fibrosis in several experimental models (34,60–63). In this study, the renal response to UUO was characterized by an increase in the expression of all four HGF mRNA transcripts. The predominant 2.3-kb transcript was significantly higher in the uPAR+/+ than the uPAR-/- mice. HGF protein deposited in the interstitium of the obstructed kidneys and may have contributed to the dampened fibrogenic response observed in the uPAR wild-type mice given that levels of the active HGF alpha chain were higher in the obstructed wild-type kidneys.

The other known uPAR ligands, high–molecular weight kininogen (64) and vitronectin, are also increased in the kidney in response to ureteral obstruction. While not investigated in this study, it is possible that uPAR-kininogen interactions also mediate renoprotective effects, as kallikrein gene therapy has been reported to attenuate glomerulosclerosis in the remnant kidney model, a therapy that was associated with increased urinary kinin excretion (65). High–molecular weight kininogen may indirectly promote the activation of uPAR-bound prourokinase, as it serves as a prekallikrein receptor, thereby juxtaposing latent uPA and one of its activators at cellular surfaces (66).

In addition to its ability to amplify uPA activity, uPAR may modulate cellular behavior (12,67,68). Although uPAR itself is attached to the plasma membrane via a GPI anchor that lacks an intracellular domain, uPAR can dimerize with other cellular receptors to initiate numerous cell surface and intracellular events. Most relevant to the present study, uPAR interacts with the scavenger receptor LDL receptor-related protein (LRP) (69). This interaction appears to be the primary pathway for clearance of extracellular PAI-1 after it binds to uPAR-uPA (5). In the present study, renal interstitial cells were shown to express LRP protein by immunostaining and the area stained was reduced in the uPAR-/- mice. The uPAR is also expressed by several interstitial cells in the wild-type mice, indicating that the uPAR-LRP scavenger receptor complex is co-expressed by renal interstitial cells and is theoretically available for PAI-1 clearance. Increased PAI-1 accumulation could explain why tPA activity, as well as uPA activity, was significantly reduced in the uPAR-/- mice.

It is also noteworthy that LRP is an endocytosis receptor for multiple ligands, including fibronectin (70), and has recently been identified as the receptor for connective tissue growth factor (71), both important components of the fibrogenic response. uPAR is also known to dimerize with members of the integrin superfamilies ({beta}1, {beta}3, and {beta}5), interactions that may trigger intracellular signaling responses and promote cell adhesion and migration. Given that uPA and its receptor are also strongly expressed by tubular cells during UUO, it is possible that uPA-uPAR-dependent cellular signaling responses may promote tubular survival within a fibrosing environment. Although the present study does not prove that the observed increase in plasminogen activator activity is the major reason for the less aggressive fibrogenic response observed in the uPAR wild-type mice, it is noteworthy that the levels of increased expression of the genes encoding the interstitial matrix proteins fibronectin and procollagen I were similar in the wild-type and knockout mice, suggesting that altered rates of matrix synthesis do not explain the differences. In contrast with the findings in this study, deficiency of uPAR failed to alter the degree of pulmonary interstitial fibrosis in mice treated with bleomycin (57). However, in that model tPA may be a more important plasminogen activator as fibrin (the primary tPA substrate) accumulation is significant and may play an important pathogenetic role, although data on the latter point are conflicting (10,72–75). It may also turn out that plasmin activity is more important in the lung than it is in the kidney on the basis of the initial studies using plasminogen-deficient mice. Nonetheless, these findings suggest that uPA-based therapies may represent a novel therapeutic approach for progressive renal disease. In fact, uPA therapy administered to animals with bleomycin-induced lung disease using adenoviral gene vectors or recombinant protein, has been reported to reduce fibrosis (76,77). Although the results of uPA therapy in chronic tubulointerstitial disease have not yet been reported, recombinant tPA treatment has been reported to reduce the extent of glomerular matrix accumulation in rats with Thy-1 nephritis (78).

In summary, the results of the present study suggest that within the kidney uPAR helps to regulate the intensity of the fibrogenic response to chronic damage, at least in part by increasing the activity of plasminogen-activating proteases. Whether polymorphisms in the uPAR gene (79) correlate with the risk of renal disease progression and whether therapeutic interventions designed to enhance uPAR expression and activity can be protective are questions deserving of future investigation.


   Acknowledgments
 
This work was funded by grant support from the National Institutes of Health DK54500 (AAE), DK58925 (JL-G), DK47659 (CEA), DK61408 (YL), and the Northwest Kidney Foundation (JL-G).


   Footnotes
 
Dr. Eric Rondeau served as Guest Editor and supervised the review and final disposition of this manuscript.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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Received for publication August 12, 2002. Accepted for publication January 31, 2003.




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