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J Am Soc Nephrol 12:736-748, 2001
© 2001 American Society of Nephrology

TIMP-1 Deficiency Does Not Attenuate Interstitial Fibrosis in Obstructive Nephropathy

HEUNGSOO KIM*, TAKASHI ODA*, JESÚS LÓPEZ-GUISA*, DIANE WING*, DYLAN R. EDWARDS{dagger}, PAUL D. SOLOWAY{ddagger} and ALLISON A. EDDY*

* The Children's Hospital and Regional Medical Center, University of Washington, Seattle, Washington
{dagger} School of Biological Sciences, University of East Anglia, Norwich, England
{ddagger} Roswell Park Cancer Institute, Department of Molecular and Cellular Biology, Buffalo, New York.

Correspondence to Dr. Allison Eddy, The Children's Hospital and Regional Medical Center, Division of Nephrology, Mail Stop CH-46, 4800 Sand Point Way NE, Seattle, WA 98105. Phone: 206-526-2524; Fax: 206-528-2636; E-mail: aeddy{at}u.washington.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Progressive renal disease as a result of renal fibrosis is caused in part by an impairment of the proteolytic machinery that normally regulates matrix turnover. The goal of the present study was to determine whether genetic deficiency of tissue inhibitor of metalloproteinases-1 (TIMP-1) could attenuate interstitial fibrosis caused by unilateral ureteral obstruction (UUO). Groups of wild-type (Timp-1) mice and TIMP-1—deficient (timp-1) mice were killed after 3 and 14 d of UUO or sham operation. Timp-1 mRNA levels were significantly increased 37- and 19-fold in the wild-type mice 3 and 14 d, respectively, after UUO operation. Matrix metalloproteinase-9 (MMP-9) activity fell in all UUO groups but remained significantly higher in the timp-1 group compared with the Timp-1 group. The degree of interstitial fibrosis (kidney collagen content and percentage of tubulointerstitial area stained with picrosirius red and collagen III) was significantly increased 14 d after UUO operation, but there was no difference between the Timp-1 and timp-1 groups. Many features of the fibrogenic response were similar between the Timp-1 and timp-1 groups, including the number of myofibroblasts and the induction of genes encoding procollagen III, fibronectin, and transforming growth factor-ß. After UUO operation, renal mRNA levels for Timp-3 and plasminogen activator inhibitor-1 were significantly higher in the TIMP-1—deficient mice. The results of this study show that elimination of TIMP-1 alone does not alter the severity of interstitial fibrosis. These findings may be due to compensation by other protease inhibitors such as TIMP-2, TIMP-3, and/or plasminogen activator inhibitor-1 or to the possibility that inhibition of intrinsic MMP activity does not constitute a profibrogenic event in the kidney.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extracellular matrix accumulation in glomeruli and the tubulointerstitium, recognized as fibrosis on histologic examination, is the pathologic hallmark of progressive renal injury. Regardless of the primary cause of the renal disease, decreased renal function correlates most closely with pathologic changes in the tubulointerstitium: interstitial fibrosis, tubular atrophy, and loss of peritubular capillaries (1). There is evidence to suggest that destructive fibrosis within the tubulointerstitium is a consequence of increased matrix protein synthesis coupled with an impairment of the pathways that normally regulate matrix remodeling and degradation. What remains unclear is the relative contribution of impaired matrix turnover to the interstitial fibrogenic response and whether interventions designed to enhance matrix degradation can significantly reduce matrix deposition in the renal interstitium. Since Gonzalez-Avila et al. (2) first reported impaired collagenolytic activity as the predominant renal metabolic abnormality associated with interstitial fibrosis in two experimental models, several studies have identified the tissue inhibitor of metalloproteinases-1 (TIMP-1) as a potential mediator of this response.

Matrix degradation is a complex and tightly regulated process that involves the interaction of numerous enzymatic cascades (3). Among the four families of the connective tissue proteases, the metalloproteinases and the serine proteases are of current interest for their possible role in the pathogenesis of renal fibrosis (4,5). The family of zinc-dependent metalloproteinases consists of several known extracellular enzymes that are classically subdivided into three groups on the basis of their domain structure and substrate preference: interstitial collagenases, stromelysins, and gelatinases. The metalloproteinases are initially secreted as proenzymes that are proteolytically activated. Enzyme activity can be blocked through interactions with one of the four known tissue inhibitors of metalloproteinases: TIMP-1, -2, -3, and -4. TIMP-2 and TIMP-3 are normally synthesized in the kidney, and relatively modest changes in their expression levels have been reported in experimental models of renal fibrosis (6). In contrast, the abundance of Timp-1 mRNA is low in normal kidneys but increases significantly in most experimental models and several human renal diseases that demonstrate fibrosis (7,8,9,10,11,12,13,14,15). Whether TIMP-1 contributes to interstitial matrix accumulation remains to be proved. In addition to its ability to inhibit metalloproteinase-dependent matrix degradation, TIMP-1 has multiple other functions, including effects on cell growth and differentiation, apoptosis, angiogenesis, stimulation of gonadal steroidogenesis, and inhibition of smooth muscle cell migration (16).

The goal of the present study was to evaluate the role of TIMP-1 in the pathogenesis of renal fibrosis by evaluating the effect of genetic TIMP-1 deficiency in a murine model of unilateral ureteral obstruction (UUO). We recently reported that TIMP-1 deficiency failed to alter the severity of interstitial fibrosis in mice with mild interstitial fibrosis induced by protein-overload proteinuria (17). The present study was undertaken to determine whether these results would be reproduced in an experimental kidney disease model characterized by aggressive scarring and renal destruction (18,19). The obstructive uropathy models also provide an opportunity to evaluate the molecular basis of renal tubulointerstitial fibrosis in the absence of proteinuria, hyperlipidemia, and primary immunologic events, each of which may modulate the fibrogenic response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
Study mice were bred in our animal facility and allowed to grow to a minimum weight of 20 g before the study began. The Timp-1—deficient (20) and wild-type control mice had an identical 129SvJae genetic background. The Timp-1 gene is located on the X-chromosome (21). All studies were performed in male mice. The genotype of the mice was confirmed by PCR analysis of DNA extracted from ear punches using two pairs of primers. The first pair amplified the neomycin resistance gene in the Timp-1 mutant allele to yield a 477-bp nucleotide product, and the second pair amplified a 394-nucleotide product in the wild-type allele. Henceforth, the wild-type mice are referred to as Timp-1 mice, indicating that they express the wild-type allele, whereas the TIMP-1—deficient mice are referred to as timp-1 mice, indicating that they express the mutant allele.

Four groups of weight-matched Timp-1—deficient and wild-type mice were studied: 3 d after UUO operation (n = 8 each), 14 d after UUO operation (n = 8 each), and both 3 and 14 d after sham operation (n = 8 each). UUO operation was performed under general anesthesia with isoflurane (Fort Dodge Animal Health, Fort Dodge, IA). The left ureter was ligated with 4.0 silk at two separate locations in the UUO group. All mice were killed by exsanguination under general anesthesia. All procedures were performed in compliance with the guidelines established by the National Research Council Guide for the Care and Use of Laboratory Animals.

Kidney Tissue Preparation
After exsanguination, the left kidney was harvested and the capsule was removed. The kidney was weighed immediately (wet weight) and then cut in half by sagittal section and divided as follows: one third of the first half kidney for paraffin section and two thirds of the halved kidney for cryostat sectioning; one fourth of the remaining half kidney for the total collagen measurement and three fourths of the second halved kidney for the total RNA (n = 5 per group) or zymography (n = 3 per group). Pieces to be embedded in paraffin section were fixed in 10% buffered formalin, and pieces for cryostat sectioning were imbedded in Tissue-Tek OCT compound (Sakura Finetek, Torrence, CA) and snap-frozen in prechilled 2-methylbutane. Pieces for zymography, mRNA extraction, and total collagen assay were snap-frozen in liquid nitrogen and stored at -80°C for subsequent use.

Gene Expression Studies
Total kidney RNA was isolated by the phenol and guanidine isothiocyanate extraction method described by Chomczynski and Sacchi (22). Total kidney RNA (4.3 or 18 µg) from each individual animal was loaded into a 1.0% agarose formaldehyde gel and separated by electrophoresis. A photomicrograph of the ethidium bromide—stained gel was obtained to evaluate RNA loading equality, then the RNA was transferred to a hybridization membrane (GeneScreen Plus, New England Nuclear Life Science Products, Boston, MA) and fixed by ultraviolet cross-linking (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 membranes were hybridized with the radiolabeled cDNA probes using the QuickHyb hybridization solution (Stratagene, La Jolla, CA). Autoradiographs were obtained and the density of each band was quantified using the NIH Image program. The density of the 28-s ribosomal bands in the formaldehyde gels were also quantified, and the results were used to adjust for any RNA-loading inequality.

cDNA Probes
The cDNA probes used were murine MMP-2 and MMP- 9 (23,24), human TIMP-1 (25), murine TIMP-2, -3, and -4 (provided by Dr. K. Leco, Ontario Cancer Institute, Toronto, Ontario, Canada) (26,27,28), murine {alpha}1(III) procollagen (provided by Dr. S. Thorgeirsson, National Cancer Institute, Bethesda, MD) (29), rat fibronectin {lambda}-rlf-1 (provided by Dr. R. Hynes, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA) (30), rat transforming growth factor-ß1 (TGF-ß1; provided by Dr. S. W. Qian, National Cancer Institute) (31), and rat plasminogen activator inhibitor-1 (PAI-1; provided by Dr. T. D. Gelehrter, University of Michigan, Ann Arbor, MI) (32).

Evaluation of Metalloproteinase Activity by Gel Zymography
Gelatin Zymography. Gelatin zymography was performed according to the method reported by Kenagy et al. (33) to analyze gelatinolytic activity. In brief, pieces of kidney that had been stored at -80°C were individually ground into a fine powder using a mortar and pestle that had been prechilled with dry ice. 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]) (34). The samples were vortexed and dialyzed using dialysis membrane Spectra/Por 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. The samples were centrifuged for 5 min (14,000 x g), and the supernatant was aliquoted after protein concentration measurement using the Bradford protein assay (Bio-Rad, Hercules, CA). The aliquoted samples were stored at -70°C until analyzed. Samples (10 µg/well) were loaded without heating onto a 7% acryl/Bis, 10% sodium dodecyl sulfate (SDS) polyacrylamide gel containing 1 mg/ml porcine skin gelatin (Sigma Chemical Company, St. Louis, MO) as substrate. Molecular markers and human MMP-2 and -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. The gel was photographed, and the size of each lytic band was measured using the NIH image analysis program.

Casein Zymography. Casein zymography was also performed to evaluate the difference in stromelysin-1 (MMP-3) activity (35) between the TIMP-1—deficient and wild-type groups as TIMP-1 also inhibits stromelysin-1 activity (36). The procedure used was identical to gelatin zymography except that the gel zymogram was made of 12% acryl/Bis, 10% SDS polyacrylamide gel, and 1 mg/ml {alpha}-casein (Sigma Chemical Co.) as substrate.

Reverse Zymography. Samples were analyzed for the presence of TIMP essentially as described by Edwards et al. (37). Briefly, samples prepared as described above were electrophoresed on non-denaturing 0.1% SDS, 12% polyacrylamide gels containing 1 mg/ml gelatin and conditioned media from BHK TK 21, which was added to 6.7% (vol/vol) as a source of gelatin-degrading enzyme. Electrophoresis was carried out at 4°C after which the gel was washed at room temperature in a solution of 2.5% Triton X 100, 50 mM Tris-Cl (pH 7.5), and 5 mM CaCl2 once for 15 min, then again overnight. The next day, the gel was rinsed once in water and incubated in 50 mM Tris-Cl (pH 7.5) and 5 mM CaCl2 for 24 h at 37°C and stained with Coomassie blue.

Western Blot Analysis
Samples were prepared as described for zymography. Samples (20 µg total protein) were separated by 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a nylon membrane, and the immunoreactive protein was visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The primary antibodies used were rabbit anti-human MMP-9 antiserum (Chemicon International Inc.) and murine anti-bovine TIMP-1 monoclonal antibody (Oncogene Research Products, Cambridge, MA). Secondary antibodies were horseradish peroxidase—conjugated goat anti-rabbit IgG antiserum (Chemicon International Inc.) and peroxidase-conjugated goat anti-mouse IgG antiserum (Sigma Chemical Co.).

Histologic Analysis of Tubulointerstitial Inflammation and Fibrosis
Immunostaining. Sections of frozen or paraffin-embedded renal tissue 4 µm thick were stained with antibodies to fibronectin (goat anti-human fibronectin; Accurate Chemical & Scientific Corp., Westbury, NY), collagen III (goat anti-human type III collagen; Southern Biotechnology Associates Inc., Birmingham, AL), mouse macrophages (rat anti-mouse F4/80 monoclonal antibody; Serotec Ltd., Oxford, UK), myofibroblasts (horseradish peroxidase—conjugated mouse anti-human smooth muscle actin monoclonal antibody; DAKO Corp., Carpinteria, CA), and TIMP-1 (murine monoclonal antibody to bovine dental pulp TIMP-1; Oncogene Research Products). The secondary antisera used were FITC-conjugated goat anti-rat IgG (Organon Teknika Corp., West Chester, PA), FITC-conjugated rabbit anti-goat IgG (Southern Biotechnology Associates), and FITC-conjugated goat anti-mouse IgG (Zymed Laboratories Inc., South San Francisco, CA).

The area (expressed as percentage of total tubulointerstitial area) of the tubulointerstitium occupied by extracellular matrix proteins was measured by computerized image analysis using the Optimas program version 6.5 (Optimas Corp., Bothell, WA). In brief, randomly selected cortical interstitial fields at x400 magnification (n = 6) from each animal were photographed using a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). The captured image was converted into a binary image and modified to eliminate areas occupied by glomeruli and vessels. The region of positive staining was automatically measured by the Optimas macro program using previously determined threshold settings. The results were expressed as percentage of positive area.

The number of F4/80-positive interstitial cells was counted manually in six random cortical fields (x400) from each experimental animal using a 10 x 10 eyepiece grid. The use of p-phenylenediamine-PBS-glycerol media containing ethidium bromide to mount coverslips facilitated the identification of interstitial cells as previously reported (38).

Picrosirius Red Staining. Picrosirius red staining was performed to evaluate the area occupied by collagen fibrils. Paraffin sections 4 µm thick were baked at 55°C for 1 h, deparaffinized, and hydrated. Sections were incubated in picrosirius red solution (1% Sirius red in saturated picric acid) for 18 h. This was followed by 0.01 N HCl treatment for 2 min, dehydration, and coverslip mounting with cytoseal 60 (Richard Allan Scientific, Kalamazoo, MI). Sections were examined by polarized light microscopy. Photographs of six random cortical fields (x400) from each animal were taken using the SPOT camera and the percentage of positive tubulointerstitial area was measured using the Optimas program.

TUNEL Assay of Apoptotic Nuclei. Apoptotic cells were detected in kidney sections using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. To distinguish between proximal and distal tubules, we performed Fx1A/TUNEL double immunostaining as described by Hughes and Johnson (39). The Fx1A antibody that reacts with proximal tubular brush border antigens was a kind gift from Dr. William Couser (University of Washington, Seattle, WA). Cells were regarded as TUNEL positive if their nuclei both were stained and had an apoptotic morphology characterized by typical nuclear pyknosis and chromatin condensation. The number of TUNEL-positive cells in each specimen was calculated in a blinded fashion by counting the number of TUNEL-positive tubular and interstitial cells separately in an average of 12 sequentially selected nonoverlapping fields of renal cortex at x400 magnification. Results were expressed as the mean number of apoptotic cells per x400 field.

Total Kidney Collagen Content
Total renal collagen was measured biochemically as described previously (40). In brief, an accurately weighed portion of the obstructed kidney was homogenized in distilled water using a pellet pestle (Kontes Scientific Glassware/Instruments, Vineland, NJ). Homogenates were hydrolyzed in 10 N HCl by incubation at 110°C for 18 h. The hydrolysate was dried by speed vacuum centrifugation over 3 to 5 h and redissolved in buffer (25 g of citric acid, 6 ml of glacial acetic acid, 60 g of sodium acetate, 17 g of sodium hydroxide in 500 ml [pH 6.0]). Total hydroxyproline in this hydrolysate was determined according to the chemical method technique of Kivirikko et al. (41). 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 weight.

Statistical Analyses
All values are expressed as mean ± 1 SD, unless otherwise stated. Results were analyzed by the Mann Whitney U test using the SPSS program (SPSS, Inc. Cary, NC). P<0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kidney and Body Weights
The mean preoperative weights of the mice were similar in all experimental groups (Table 1). Most of the mice experienced a small weight reduction in the postoperative period. The wet weight of the experimental left kidney was significantly increased 3 d (183 ± 34 and 186 ± 27 for Timp-1 and timp-1, respectively) and decreased 14 d (93 ± 10 and 116 ± 26) after UUO operation compared with the wet kidney weight of the sham-operated kidneys (138 ± 18 and 143 ± 15). Fourteen d after UUO operation, the mean kidney weight of the Timp-1 group was less than the timp-1 group, but the difference was not statistically significant (P = 0.052).


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  		Table 1. Body weight
 

Renal Expression of Genes Encoding Gelatinases and TIMP
Northern blot analysis of total kidney RNA from wild-type mice identified a faint Timp-1 mRNA band in the sham-operated mice and a significant increase in Timp-1 mRNA levels 3 and 14 d after UUO operation (Figure 1). A very faint band was identified in the timp-1 mice consistent with previous reports and thought to represent an unstable timp-1 mutant transcript (20).



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  		Figure 1. Timp-1 Northern blot autoradiograph (A) and graph of mean band density (B). Results are expressed as mean fold increase ± 1 SD with the baseline value defined as the density of the weakest band on the Northern blot. The 28-s band from the ethidium bromide—stained gel is illustrated below each lane. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1—deficient mice expressing the mutant allele. *, P<0.05 compared with sham group of same genotype; **, P<0.05 versus day 3 Timp-1 UUO group; ***, P<0.05 versus day 14 Timp-1 UUO group.

 

The possibility that deficiency of TIMP-1 resulted in compensation by increased expression of other protease inhibitors was evaluated. Northern blot analysis using a Timp-2 cDNA probe identified two transcripts of 3.5 kb and 1.0 kb. Compared with sham-operated kidneys, both transcripts were significantly increased 3 and 14 d after UUO operation with the single exception of the 3.5-kb transcript in the day 3 UUO timp-1 group (Figure 2, A and B). In comparing the response to UUO between the Timp-1 and timp-1 groups, there was no consistent difference. The 1.0-kb Timp-2 transcript was significantly higher in the timp-1 group at 3 d, but it was significantly lower on day 14.



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  		Figure 2. Northern blot analysis of renal protease inhibitors Timp-2 and Timp-3. (A and C) autoradiographs of the Northern blot with the ethidium bromide—stained 28-s ribosomal band illustrated below each lane. (B and D) Results of analysis of the band density expressed as mean fold increase ± 1 SD with the baseline value defined as the density of the weakest band on the Northern blot. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1—deficient mice expressing the mutant allele. *, P<0.05 compared with sham group of same genotype; **, P<0.05 versus day 3 Timp-1 UUO group; ***, P<0.05 versus day 14 Timp-1 UUO group.

 

Northern blot analysis using a Timp-3 cDNA probe identified 4.5-kb and 2.4-kb transcripts. On day 14 after UUO operation, there was a significant decrease in both transcripts compared with the sham-operation groups (Figure 2, C and D). The 4.5-kb Timp-3 was significantly higher 14 d after UUO operation in the timp-1 group compared with the Timp-1 group. A Timp-4 transcript was not detected in total RNA isolated from any of the experimental groups (data not shown).

Compared with genetically identical sham groups, PAI-1 mRNA levels were significantly increased in the Timp-1 group 14 d after UUO operation and at both 3 and 14 d in the timp-1 group. At both 3 and 14 d, PAI-1 mRNA levels were significantly higher in the timp-1 group compared with the Timp-1 group. PAI-1 mRNA results for the Timp-1 and timp-1 groups, respectively, expressed as fold increase relative to the lowest mean value were as follows: 3 d sham, 1.5 ± 0.1 versus 1.0 ± 0.4; 3 d UUO, 1.7 ± 0.4 versus 2.8 ± 0.4; 14 d sham, 1.0 ± 0.2 versus 1.1 ± 0.5; and 14 d UUO, 2.8 ± 0.2 versus 3.7 ± 0.6.

Renal MMP-9 mRNA levels were significantly increased in both UUO groups compared with the genetically identical sham-operated control groups on days 3 and 14. No significant differences were observed between Timp-1 and timp-1 UUO groups. Results for the Timp-1 and timp-1 groups, respectively, expressed as fold increase relative to the lowest mean value were as follows: 3 d sham, 1.2 ± 0.4 versus 2.1 ± 0.9; 3 d UUO, 3.6 ± 1.2 versus 3.3 ± 0.5; 14 d sham, 1.0 ± 0.3 versus 1.5 ± 0.2; and 14 d UUO, 3.7 ± 1.4 versus 3.4 ± 0.6.

Hybridization using a MMP-2 cDNA probe identified two transcripts of 4.0 kb and 3.2 kb. Renal mRNA levels of both MMP-2 transcripts were significantly increased in the day 3 and day 14 UUO groups compared with their sham-operated control groups. There was no significant differences in the 4.0-kb transcript between the Timp-1 and the timp-1 groups at 3 and 14 d. Results for the 4.0-kb transcript in the Timp-1 and timp-1 groups, respectively, expressed as fold increase relative to the lowest mean value were as follows: 3 d sham, 1.1 ± 0.3 versus 1.0 ± 0.5; 3 d UUO, 2.6 ± 0.6 versus 2.8 ± 0.6; 14 d sham, 1.5 ± 0.5 versus 1.0 ± 0.7; and 14 d UUO, 8.1 ± 1.5 versus 6.7 ± 1.1. In contrast, at 14 d, the 3.2-kb transcript mRNA levels were significantly higher in the Timp-1 group compared with the timp-1 group (24.2 ± 1.1 versus 20.2 ± 1.3-fold increase).

Renal Metalloproteinase Activity
Gelatin zymography detected strong lytic bands representing MMP-9 activity and considerably smaller bands due to MMP-2 activity. After UUO operation, renal MMP-9 activity was decreased at 14 d in the Timp-1 group and at 3 and 14 d in the timp-1 group compared with their sham control groups (P = 0.05; n = 3 per group; Figure 3). However, basal MMP-9 activity in the sham kidneys was higher in the timp-1 kidneys, and 3 and 14 d after UUO operation, mean MMP-9 activity was higher in the timp-1 group compared with the Timp-1 group (P = 0.05). In contrast, UUO induced MMP-2 activity, and there was no difference between the Timp-1 and the timp-1 groups. Clear bands of renal caseinolytic activity were not detected in any of the experimental groups (data not shown).



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  		Figure 3. Gelatin gel zymography illustrating renal MMP-9 and MMP-2 activity (A). The density of the lytic bands expressed as mean arbitrary units ± 1 SD is summarized in B. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1-deficient mice expressing the mutant allele. *, P = 0.05 compared with sham group of same genotype (n = 3 per group); **, P = 0.05 versus day 3 Timp-1 UUO group (n = 3 per group); ***, P = 0.05 versus day 14 Timp-1 UUO group (n = 3 per group).

 

MMP-9 Western Blotting
To investigate further the discrepancy between increased MMP-9 mRNA levels and decreased MMP-9 gelatinase activity, we evaluated MMP-9 protein levels by Western blotting. The findings were consistent with the results of the gelatin zymography showing decreased MMP-9 protein in the UUO groups compared with the sham groups (Figure 4).



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  		Figure 4. MMP-9 Western blot of kidney extracts. Each lane was loaded with 20 µg of total kidney protein. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1-deficient mice expressing the mutant allele.

 

TIMP-1 Protein and Activity
Western blotting and reverse zymography were unsuccessful in detecting convincing bands corresponding to TIMP-1 protein and inhibitory gelatinolytic activity, respectively, within kidney extracts (data not shown).

Severity of Renal Fibrosis
Total kidney collagen was significantly increased 14 d after UUO compared with sham-operated kidneys, but there was no difference found between the Timp-1 and the timp-1 groups (Figure 5). Significant interstitial fibrosis was confirmed histologically by immunofluorescence staining for collagen III (Figure 6) with no significant differences between the Timp-1 and the timp-1 groups. Similar findings were observed when the severity of fibrosis was evaluated by picrosirius red staining. Results for the Timp-1 and timp-1 groups, respectively, expressed as percentage of tubulointerstitial area stained were as follows: 3 d sham, 0.2 ± 0.2 versus 0.3 ± 0.1; 3 d UUO, 0.5 ± 0.4 versus 0.6 ± 0.3; 14 d sham, 0.2 ± 0.1 versus 0.5 ± 0.3; and 14 d UUO, 4.4 ± 1.1 versus 4.2 ± 1.8.



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  		Figure 5. Total kidney collagen. Results are mean µg collagen/mg kidney wet weight ± 1 SD. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1-deficient mice expressing the mutant allele. *, P < 0.05 compared with sham group of same genotype.

 


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  		Figure 6. (A) Photomicrographs of interstitial collagen III deposition detected by indirect immunofluorescence staining. The percentage of tubulointerstitial area stained expressed as mean ± 1 SD is illustrated in B. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1-deficient mice expressing the mutant allele. *, P < 0.05 compared with sham group of same genotype. Magnification, x400.

 

TGF-ß and Matrix Protein Gene Expression
Northern blot analysis confirmed that there was no difference between Timp-1 and timp-1 mice with respect to the induction of genes encoding TGF-ß and the matrix genes {alpha}1(III) procollagen and fibronectin in response to UUO. Compared with sham-operated kidney, renal mRNA levels for these genes were significantly increased 3 d (procollagen III, 26.3 ± 4.5 versus 26.3 ± 3.6; fibronectin, 2336 ± 907 versus 2488 ± 873; TGF-ß, 5.0 ± 0.6 versus 5.5 ± 0.7-fold increase above lowest mean sham group value) and 14 d (procollagen III, 29.6 ± 6.5 versus 29.0 ± 3.0; fibronectin, 3341 ± 1586 versus 3281 ± 980; TGF-ß, 5.8 ± 1.2 versus 5.8 ± 0.7-fold increase) after UUO operation.

Interstitial Myofibroblasts, Macrophages, and Tubular Apoptotic Cells
The number of {alpha}-smooth muscle actin—positive interstitial myofibroblasts was significantly increased 3 and 14 d after UUO operation and was similar in intensity between the Timp-1 and timp-1 groups (Figure 7). A significant interstitial infiltrate of F4/80-positive interstitial macrophages developed in response to UUO and was similar in the Timp-1 and timp-1 groups at day 14 (Figure 8). This interstitial infiltrate appeared more rapidly (day 3) in the timp-1 group but was similar in intensity to the Timp-1 UUO group on day 14. The numbers of TUNEL-positive interstitial and proximal tubule cells were significantly increased 3 and 14 d after UUO operation (Figure 9). The number of apoptotic interstitial cells was significantly higher in the timp-1 UUO groups compared with the Timp-1 UUO groups. There was no difference in the number of apoptotic proximal tubule cells. Results for the Timp-1 and timp-1 groups, respectively, expressed as the number of TUNEL-positive tubular cells per x400 field were as follows: 3 d sham, 0 ± 0 versus 0 ± 0; 3 d UUO, 0.07 ± 0.08 versus 0.09 ± 0.08; 14 d sham, 0 ± 0 versus 0.02 ± 0.04; and 14 d UUO, 0.3 ± 0.2 versus 0.5 ± 0.2. Very few TUNEL-positive distal tubule cells were observed after UUO (data not shown).



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  		Figure 7. (A) Photomicrographs of interstitial myofibroblasts detected by immunohistochemical staining for {alpha}-smooth muscle actin. The percentage of tubulointerstitial area stained expressed as mean ± 1 SD is illustrated in B. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1-deficient mice expressing the mutant allele. *, P < 0.05 compared with sham group of same genotype. Magnification, x400.

 


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  		Figure 8 (A) Photomicrographs of interstitial monocytes/macrophages detected by indirect immunofluorescence staining for the F4/80 antigen. The number of positive cells per x400 field expressed as mean ± 1 SD is illustrated in B. Timp-1, wild-type mice expressing the normal allele; timp-1, TIMP-1-deficient mice expressing the mutant allele. *, P < 0.05 compared with sham group of same genotype; **, P < 0.05 versus day 3 Timp-1 UUO group. Magnification, x400.

 


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  		Figure 9. TUNEL-positive tubulointerstitial cells. Photomicrographs illustrate (arrows) a positive interstitial cell (A) and a positive proximal tubule cell (B) in a mouse with UUO. Proximal tubular cells are indicated by the dark brush border staining reaction with the anti-Fx1A antibody. The number of TUNEL-positive interstitial cells (C) is expressed as mean number of positive cells per x400 field ± 1 SD. Magnification, x630 in A and B.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study detected a striking 20- to 40-fold increase in renal Timp-1 mRNA levels in mice in response to ureteral obstruction, a finding that has been reported previously in rats (13) and rabbits (12). In fact, upregulated expression of Timp-1 mRNA has been observed as an almost universal feature of solid organ fibrosis, be it in the kidney (6), lung (42), or liver (43). A temporal association has been made between normalization of Timp-1 mRNA levels and reversal of hepatic fibrosis in a rat model of CCl4-induced liver fibrosis, suggesting a relationship between TIMP-1-dependent inhibition of collagen turnover and liver fibrosis (43). Despite the numerous associations between TIMP-1 and fibrotic states, definitive proof that TIMP-1 plays a significant pathogenetic role is still lacking. The results of the present study demonstrate clearly that genetic deficiency of TIMP-1 fails to attenuate the severity of renal fibrosis after UUO. These results are consistent with our recent findings in a much less aggressive model of renal interstitial fibrosis induced by protein-overload proteinuria (17).

The most likely explanation for these findings is genetic redundancy with compensation for a TIMP-1-deficient state by another MMP inhibitor. Likely candidates include TIMP-2 and TIMP-3. TIMP-2 is constitutively expressed in the kidney. In response to UUO, 2- to 14-fold increases in Timp-2 mRNA were observed. Although there was no difference in the Timp-2 response between the TIMP-1-deficient and wild-type mice, it is possible that this inhibitor was present in sufficient quantity to block MMP activity to a similar degree in both lines of mice. In contrast, Timp-3 is abundantly expressed in normal kidney, and its expression was significantly reduced after UUO. However, in comparing the response in the TIMP-1-deficient and wild-type mice, Timp-3 mRNA levels were significantly higher at both 3 and 14 d after UUO operation in the TIMP-1-deficient mice. TIMP-3 is unique among the known MMP inhibitors because of its affinity for and exclusive localization within extracellular matrix (16). It may be especially effective as an MMP inhibitor at extracellular sites of active fibrosis.

Changes in PAI-1 expression were unexpected and deserve further investigation. As reported in several studies, induction of PAI-1 gene expression is frequently observed in fibrotic disease states (6). This serine protease inhibitor is thought to have profibrotic effects as a consequence of decreased generation of plasmin. Plasmin is known to be a potent activator of prometalloproteinases in vitro, and a similar role in vivo has been proposed (44). Renal PAI-1 mRNA levels were significantly higher in the TIMP-1-deficient mice at both 3 and 14 d after UUO operation. We recently found that the severity of renal fibrosis is significantly reduced in PAI-1-deficient mice compared with wild-type mice after UUO operation (Oda et al., manuscript submitted), raising the distinct possibility that these differences in PAI-1 expression are relevant to the findings in the present study.

An important question is whether the differences in TIMP-1 expression were associated with functional differences in renal MMP activity. This study focused on gelatinolytic activity for two reasons. First, gelatinase activity is the strongest MMP activity currently recognized in the kidney. In fact, when the gel substrate was changed from gelatin to casein in the present study, convincing lytic bands were barely visible. Second, TIMP-1 has high specificity for MMP-9, and it also binds to pro-MMP-9 (16). In addition to gelatin, MMP-9 (92-kD type IV collagenase or gelatinase B) also degrades collagens type IV and V, fibronectin, laminin, entactin, elastin, and proteoglycans; MMP-2 (72-kD type IV collagenase or gelatinase A) also degrades collagens type IV, V, VII, X, and XI, fibronectin, laminin, entactin, elastin, and proteoglycans (45,46). However, several additional members of the MMP family may be expressed in the kidney and inhibited by TIMP-1, Of particular note is that gelatinases do not degrade fibrillar collagens such as collagen type I and III that also accumulate in the renal interstitium during fibrosis. Also unknown is whether each of the matrix proteins that constitutes the interstitial scar is equally detrimental to renal structure and function. Once these questions are resolved, we will be better positioned to focus on the specific proteases and protease inhibitors that regulate the turnover of the critical constituents of the interstitial scar.

In the present study, a strong gelatinolytic band corresponding to MMP-9 activity was present in extracts prepared from sham kidney homogenates. This activity was significantly decreased after UUO operation. However, baseline MMP-9 activity was significantly higher in the TIMP-1-deficient mice and remained significantly higher than levels observed in the wild-type mice 3 and 14 d after UUO operation. An interesting finding was a significant increase in renal MMP-9 mRNA levels after UUO operation. Because the expression of MMP is controlled primarily at the level of transcription (47), this difference between MMP-9 mRNA and activity suggests either decreased activation of MMP-9 or inhibitor-dependent inactivation of MMP-9 activity. In data not shown, pretreatment of kidney extracts with 4-aminophenylmercuric acetate to activate latent MMP-9 did not result in a significant increase in MMP-9 activity. Furthermore, Western blotting identified a single distinct band. These findings argue against the decreased activation hypothesis and favor the conclusion that the MMP-9 activity was decreased by its inhibitors.

Renal MMP-2 activity is considerably lower than MMP-9 activity in normal kidneys. In response to UUO, both MMP-2 mRNA and activity levels were significantly increased. The degree of change was similar in the TIMP-1-deficient and wild-type mice with the single exception that the less abundant 3.2-kb transcript was significantly higher in the TIMP-1-deficient group on day 14.

Several other key aspects of the interstitial fibrogenic response were evaluated and found to be similar between the Timp-1 and timp-1 groups, indicating that the TIMP-1 deficiency did not induce other compensatory responses. In particular, the expression of TGF-ß and matrix genes {alpha}1(III) procollagen and fibronectin were similar. Infiltration of monocytes into the interstitium occurred more rapidly (day 3) in the TIMP-1-deficient group, but this difference was no longer evident on day 14. It is noteworthy that a unique feature of tubulointerstitial disease observed in our studies with PAI-1-deficient mice was a significant reduction in the number of interstitial macrophages (Oda et al., manuscript submitted). Whether the increased expression of PAI-1 in the TIMP-1-deficient mice facilitated early macrophage recruitment is speculative.

The observation that the severity of renal fibrosis was unaffected by the absence of TIMP-1 raises the possibility that TIMP-1 may be mediating other effects in the kidney. The multifunctionality of TIMP-1 is increasingly recognized (16). What this effect might be remains unknown. TIMP-1 has been reported to inhibit B-cell death by apoptosis (48). The results of the present study suggest that TIMP-1 may play a similar role in the kidney as the deficiency of TIMP-1 resulted in a significant increase in the number of apoptotic interstitial cells 3 and 14 d after UUO operation. The functional significance of this difference is unclear. TIMP-1 has also been reported to inhibit smooth muscle cell migration (49). Although the origin of the interstitial myofibroblasts that typify progressive renal disease remains controversial, in the present study the number of interstitial {alpha}-smooth muscle actin-positive interstitial cells was not altered in the TIMP-1-deficient mice with UUO, suggesting that TIMP-1-dependent inhibition of perivascular cell migration into the interstitium is not a feature of renal fibrosis.

A major limitation of the present study was the inability to localize TIMP-1 protein and to demonstrate TIMP-1 activity in the kidney. Similar difficulties were encountered in other experimental systems as well. The anti-TIMP-1 antibody used in these studies is reported to react with mouse TIMP-1, but it did not produce a staining pattern or a reaction on Western blots that was convincingly different from background results produced when the secondary antibody was used alone despite many different blocking procedures. Results from our previous studies in rats identified both tubular and interstitial cells as the intrarenal source of TIMP-1 production after injury, and we predict that the same would be true in mice (50). Reverse zymography to detect MMP inhibitor activity has proved technically challenging when efforts are made to isolate the inhibitors from tissue rather than cultured cells or their supernatant. The procedure used in the present study produced clear bands of activity when recombinant TIMP-1, -2, or -3 were used, but the only convincing activity observed in the kidney extracts corresponded to TIMP-3 activity (data not shown).

In summary, the fibrogenic response to UUO in mice includes a significant upregulation of Timp-1. However, despite genetic deficiency of TIMP-1 and higher renal MMP-9 activity, the severity of renal interstitial fibrosis was not reduced in the TIMP-1-deficient mice. Significant differences in the renal expression of TIMP-3 and PAI-1 and/or high constitutive expression of TIMP-2 may compensate when TIMP-1 is not available during the active phase of renal fibrosis. Future studies in mice that are genetically deficient in TIMP-2 and TIMP-3 and in mice that have combined deficiencies in TIMP-1 and PAI-1, TIMP-2, and/or TIMP-3 should be able to clarify the mechanism of compensation and establish the role of impaired matrix turnover in the renal fibrogenic response. However, an alternative explanation is that inhibition of intrinsic MMP activity does not represent a profibrotic event in the kidney.


   Acknowledgments
 
This work was funded by National Institutes of Health Grant No. DK54500 (A.A.E.). The authors acknowledge the outstanding technical assistance of Dr. Richard D. Kenagy with zymographic studies and Dr. Jeremy Hughes with the TUNEL assay. The assistance of Ruthanne Naranjo with reference formatting is greatly appreciated.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication June 21, 2000. Accepted for publication September 12, 2000.




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