timp-1 deficiency does not attenuate interstitial fibrosis in obstructive nephropathy

TIMP-1 Deficiency Does Not Attenuate Interstitial Fibrosis inObstructive Nephropathy

HEUNGSOO KIM,* TAKASHI ODA,* JESUS LOPEZ-GUISA,* DIANE WING,*DYLAN R. EDWARDS,† PAUL D. SOLOWAY,‡ and ALLISON A. EDDY**The Children’s Hospital and Regional Medical Center, University of Washington, Seattle, Washington;†School of Biological Sciences, University of East Anglia, Norwich, England; and‡Roswell Park CancerInstitute, Department of Molecular and Cellular Biology, Buffalo, New York.

Abstract.Progressive renal disease as a result of renal fibrosisis caused in part by an impairment of the proteolytic machinerythat normally regulates matrix turnover. The goal of the presentstudy was to determine whether genetic deficiency of tissueinhibitor of metalloproteinases-1 (TIMP-1) could attenuate in-terstitial 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 orsham operation.Timp-1 mRNA levels were significantly in-creased 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 signif-icantly higher in thetimp-1 group compared with theTimp-1group. The degree of interstitial fibrosis (kidney collagen con-tent and percentage of tubulointerstitial area stained with picro-

sirius red and collagen III) was significantly increased 14 dafter UUO operation, but there was no difference between theTimp-1 and timp-1 groups. Many features of the fibrogenicresponse were similar between theTimp-1and timp-1 groups,including the number of myofibroblasts and the induction ofgenes encoding procollagen III, fibronectin, and transforminggrowth factor-b. After UUO operation, renal mRNA levels forTimp-3 and plasminogen activator inhibitor-1 were signifi-cantly higher in the TIMP-1–deficient mice. The results of thisstudy show that elimination of TIMP-1 alone does not alter theseverity of interstitial fibrosis. These findings may be due tocompensation by other protease inhibitors such as TIMP-2,TIMP-3, and/or plasminogen activator inhibitor-1 or to thepossibility that inhibition of intrinsic MMP activity does notconstitute a profibrogenic event in the kidney.

Extracellular matrix accumulation in glomeruli and the tubu-lointerstitium, recognized as fibrosis on histologic examina-tion, is the pathologic hallmark of progressive renal injury.Regardless of the primary cause of the renal disease, decreasedrenal function correlates most closely with pathologic changesin the tubulointerstitium: interstitial fibrosis, tubular atrophy,and loss of peritubular capillaries (1). There is evidence tosuggest that destructive fibrosis within the tubulointerstitium isa consequence of increased matrix protein synthesis coupledwith an impairment of the pathways that normally regulatematrix remodeling and degradation. What remains unclear isthe relative contribution of impaired matrix turnover to theinterstitial fibrogenic response and whether interventions de-signed to enhance matrix degradation can significantly reducematrix deposition in the renal interstitium. Since Gonzalez-Avila et al.(2) first reported impaired collagenolytic activity asthe predominant renal metabolic abnormality associated with

interstitial fibrosis in two experimental models, several studieshave identified the tissue inhibitor of metalloproteinases-1(TIMP-1) as a potential mediator of this response.

Matrix degradation is a complex and tightly regulated pro-cess that involves the interaction of numerous enzymatic cas-cades (3). Among the four families of the connective tissueproteases, the metalloproteinases and the serine proteases areof current interest for their possible role in the pathogenesis ofrenal fibrosis (4,5). The family of zinc-dependent metallopro-teinases consists of several known extracellular enzymes thatare classically subdivided into three groups on the basis of theirdomain structure and substrate preference: interstitial collag-enases, stromelysins, and gelatinases. The metalloproteinasesare initially secreted as proenzymes that are proteolyticallyactivated. Enzyme activity can be blocked through interactionswith one of the four known tissue inhibitors of metalloprotein-ases: TIMP-1, -2, -3, and -4. TIMP-2 and TIMP-3 are normallysynthesized in the kidney, and relatively modest changes intheir expression levels have been reported in experimentalmodels of renal fibrosis (6). In contrast, the abundance ofTimp-1mRNA is low in normal kidneys but increases signif-icantly in most experimental models and several human renaldiseases that demonstrate fibrosis (7–15). Whether TIMP-1contributes to interstitial matrix accumulation remains to beproved. In addition to its ability to inhibit metalloproteinase-dependent matrix degradation, TIMP-1 has multiple otherfunctions, including effects on cell growth and differentiation,

Received June 21, 2000. Accepted September 12, 2000.Correspondence to Dr. Allison Eddy, The Children’s Hospital and RegionalMedical Center, Division of Nephrology, Mail Stop CH-46, 4800 Sand PointWay NE, Seattle, WA 98105. Phone: 206-526-2524; Fax: 206-528-2636;E-mail: [email protected]

1046-6673/1204-0736Journal of the American Society of NephrologyCopyright © 2001 by the American Society of Nephrology

J Am Soc Nephrol 12: 736–748, 2001

apoptosis, angiogenesis, stimulation of gonadal steroidogene-sis, and inhibition of smooth muscle cell migration (16).

The goal of the present study was to evaluate the role ofTIMP-1 in the pathogenesis of renal fibrosis by evaluating theeffect of genetic TIMP-1 deficiency in a murine model ofunilateral ureteral obstruction (UUO). We recently reportedthat TIMP-1 deficiency failed to alter the severity of interstitialfibrosis in mice with mild interstitial fibrosis induced by pro-tein-overload proteinuria (17). The present study was under-taken to determine whether these results would be reproducedin an experimental kidney disease model characterized byaggressive scarring and renal destruction (18,19). The obstruc-tive uropathy models also provide an opportunity to evaluatethe molecular basis of renal tubulointerstitial fibrosis in theabsence of proteinuria, hyperlipidemia, and primary immuno-logic events, each of which may modulate the fibrogenicresponse.

Materials and MethodsExperimental Design

Study mice were bred in our animal facility and allowed to grow toa minimum weight of 20 g before the study began. TheTimp-1–deficient (20) and wild-type control mice had an identical 129SvJae

genetic background. TheTimp-1 gene is located on the X-chromo-some (21). All studies were performed in male mice. The genotype ofthe mice was confirmed by PCR analysis of DNA extracted from earpunches using two pairs of primers. The first pair amplified theneomycin resistance gene in theTimp-1 mutant allele to yield a477-bp nucleotide product, and the second pair amplified a 394-nucleotide product in the wild-type allele. Henceforth, the wild-typemice are referred to asTimp-1mice, indicating that they express thewild-type allele, whereas the TIMP-1–deficient mice are referred to astimp-1 mice, indicating that they express the mutant allele.

Four groups of weight-matchedTimp-1–deficient and wild-typemice were studied: 3 d after UUO operation (n 5 8 each), 14 d afterUUO operation (n 5 8 each), and both 3 and 14 d after sham operation(n 5 8 each). UUO operation was performed under general anesthesiawith isoflurane (Fort Dodge Animal Health, Fort Dodge, IA). The leftureter was ligated with 4.0 silk at two separate locations in the UUOgroup. All mice were killed by exsanguination under general anes-thesia. All procedures were performed in compliance with the guide-lines established by the National Research Council Guide for the Careand Use of Laboratory Animals.

Kidney Tissue PreparationAfter exsanguination, the left kidney was harvested and the capsule

was removed. The kidney was weighed immediately (wet weight) andthen cut in half by sagittal section and divided as follows: one third ofthe first half kidney for paraffin section and two thirds of the halvedkidney for cryostat sectioning; one fourth of the remaining half kidneyfor the total collagen measurement and three fourths of the secondhalved kidney for the total RNA (n 5 5 per group) or zymography(n 5 3 per group). Pieces to be embedded in paraffin section werefixed in 10% buffered formalin, and pieces for cryostat sectioningwere imbedded in Tissue-Tek OCT compound (Sakura Finetek, Tor-rence, CA) and snap-frozen in prechilled 2-methylbutane. Pieces forzymography, mRNA extraction, and total collagen assay were snap-frozen in liquid nitrogen and stored at280°C for subsequent use.

Gene Expression StudiesTotal kidney RNA was isolated by the phenol and guanidine isothio-

cyanate extraction method described by Chomczynski and Sacchi (22).Total kidney RNA (4.3 or 18mg) from each individual animal was loadedinto a 1.0% agarose formaldehyde gel and separated by electrophoresis.A photomicrograph of the ethidium bromide–stained gel was obtained toevaluate RNA loading equality, then the RNA was transferred to ahybridization membrane (GeneScreen Plus, New England Nuclear LifeScience Products, Boston, MA) and fixed by ultraviolet cross-linking(UV Crosslinker, Hoeffer Scientific Instruments, San Francisco, CA).Complementary DNA probes were radiolabeled with32P dCTP (3000Ci/mmol) by random priming with T7 Quick Prime kit (PharmaciaBiotech, Piscataway, NJ). The membranes were hybridized with theradiolabeled cDNA probes using the QuickHyb hybridization solution(Stratagene, La Jolla, CA). Autoradiographs were obtained and the den-sity of each band was quantified using the NIH Image program. Thedensity of the 28-s ribosomal bands in the formaldehyde gels were alsoquantified, and the results were used to adjust for any RNA-loadinginequality.

cDNA ProbesThe 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) (26–28),murine a1(III) procollagen (provided by Dr. S. Thorgeirsson, Na-tional Cancer Institute, Bethesda, MD) (29), rat fibronectinl-rlf-1(provided by Dr. R. Hynes, Center for Cancer Research, Massachu-setts Institute of Technology, Cambridge, MA) (30), rat transforminggrowth factor-b1 (TGF-b1; provided by Dr. S. W. Qian, NationalCancer Institute) (31), and rat plasminogen activator inhibitor-1(PAI-1; provided by Dr. T. D. Gelehrter, University of Michigan, AnnArbor, MI) (32).

Evaluation of Metalloproteinase Activity by GelZymography

Gelatin Zymography. Gelatin zymography was performed ac-cording to the method reported by Kenagyet al. (33) to analyzegelatinolytic activity. In brief, pieces of kidney that had been stored at280°C were individually ground into a fine powder using a mortarand pestle that had been prechilled with dry ice. The powder washomogenized in extraction buffer (0.05 M Tris, 0.01 M CaCl2, 2.0 Mguanidine HCl, 0.2% Triton X-100 [pH 7.5]) (34). The samples werevortexed and dialyzed using dialysis membrane Spectra/Por 1 (Spec-trum 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 werecentrifuged for 5 min (14,0003 g), and the supernatant was aliquotedafter protein concentration measurement using the Bradford proteinassay (Bio-Rad, Hercules, CA). The aliquoted samples were stored at270°C until analyzed. Samples (10mg/well) were loaded withoutheating onto a 7% acryl/Bis, 10% sodium dodecyl sulfate (SDS)polyacrylamide gel containing 1 mg/ml porcine skin gelatin (SigmaChemical Company, St. Louis, MO) as substrate. Molecular markersand human MMP-2 and -9 standards (Chemicon International Inc.,Temecula, CA) were also loaded into the outer wells. After proteinseparation by electrophoresis, the gel was rinsed in 2.5% Triton X-100at room temperature with gentle shaking for 30 min. After incubationfor 17 to 20 h at 37°C in a solution containing 50 mM Tris and 10 mMCaCl2 (pH 7.8), the gel was stained with 0.002% Coomassie blue. Thegel was photographed, and the size of each lytic band was measuredusing the NIH image analysis program.

J Am Soc Nephrol 12: 736–748, 2001 TIMP-1 Deficiency and Interstitial Fibrosis 737

Casein Zymography. Casein zymography was also performedto evaluate the difference in stromelysin-1 (MMP-3) activity (35)between the TIMP-1–deficient and wild-type groups as TIMP-1 alsoinhibits stromelysin-1 activity (36). The procedure used was identicalto gelatin zymography except that the gel zymogram was made of12% acryl/Bis, 10% SDS polyacrylamide gel, and 1 mg/mla-casein(Sigma Chemical Co.) as substrate.

Reverse Zymography. Samples were analyzed for the presenceof TIMP essentially as described by Edwardset al. (37). Briefly,samples prepared as described above were electrophoresed on non-denaturing 0.1% SDS, 12% polyacrylamide gels containing 1 mg/mlgelatin and conditioned media from BHK TK 21, which was added to6.7% (vol/vol) as a source of gelatin-degrading enzyme. Electrophore-sis was carried out at 4°C after which the gel was washed at roomtemperature in a solution of 2.5% Triton X 100, 50 mM Tris-Cl (pH7.5), and 5 mM CaCl2 once for 15 min, then again overnight. The nextday, 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 withCoomassie blue.

Western Blot AnalysisSamples were prepared as described for zymography. Samples (20

mg total protein) were separated by 12% SDS-polyacrylamide gelelectrophoresis. Proteins were transferred to a nylon membrane, andthe immunoreactive protein was visualized using enhanced chemilu-minescence (Amersham Pharmacia Biotech Inc., Piscataway, NJ).The primary antibodies used were rabbit anti-human MMP-9 anti-serum (Chemicon International Inc.) and murine anti-bovine TIMP-1monoclonal antibody (Oncogene Research Products, Cambridge,MA). Secondary antibodies were horseradish peroxidase–conjugatedgoat anti-rabbit IgG antiserum (Chemicon International Inc.) andperoxidase-conjugated goat anti-mouse IgG antiserum (Sigma Chem-ical Co.).

Histologic Analysis of Tubulointerstitial Inflammationand Fibrosis

Immunostaining. Sections of frozen or paraffin-embedded renaltissue 4mm thick were stained with antibodies to fibronectin (goatanti-human fibronectin; Accurate Chemical & Scientific Corp., West-bury, NY), collagen III (goat anti-human type III collagen; SouthernBiotechnology Associates Inc., Birmingham, AL), mouse macro-phages (rat anti-mouse F4/80 monoclonal antibody; Serotec Ltd.,Oxford, UK), myofibroblasts (horseradish peroxidase–conjugatedmouse anti-human smooth muscle actin monoclonal antibody; DAKOCorp., Carpinteria, CA), and TIMP-1 (murine monoclonal antibody tobovine dental pulp TIMP-1; Oncogene Research Products). The sec-ondary antisera used were FITC-conjugated goat anti-rat IgG (Or-ganon Teknika Corp., West Chester, PA), FITC-conjugated rabbitanti-goat IgG (Southern Biotechnology Associates), and FITC-conju-gated goat anti-mouse IgG (Zymed Laboratories Inc., South SanFrancisco, CA).

The area (expressed as percentage of total tubulointerstitial area) ofthe tubulointerstitium occupied by extracellular matrix proteins wasmeasured by computerized image analysis using the Optimas programversion 6.5 (Optimas Corp., Bothell, WA). In brief, randomly selectedcortical interstitial fields at3400 magnification (n 5 6) from eachanimal were photographed using a SPOT digital camera (DiagnosticInstruments, Inc., Sterling Heights, MI). The captured image wasconverted into a binary image and modified to eliminate areas occu-pied by glomeruli and vessels. The region of positive staining wasautomatically measured by the Optimas macro program using previ-

ously determined threshold settings. The results were expressed aspercentage of positive area.

The number of F4/80-positive interstitial cells was counted manu-ally in six random cortical fields (3400) from each experimentalanimal using a 103 10 eyepiece grid. The use of p-phenylenedi-amine-PBS-glycerol media containing ethidium bromide to mountcoverslips facilitated the identification of interstitial cells as previ-ously reported (38).

Picrosirius Red Staining. Picrosirius red staining was per-formed to evaluate the area occupied by collagen fibrils. Paraffinsections 4mm thick were baked at 55°C for 1 h, deparaffinized, andhydrated. Sections were incubated in picrosirius red solution (1%Sirius red in saturated picric acid) for 18 h. This was followed by 0.01N HCl treatment for 2 min, dehydration, and coverslip mounting withcytoseal 60 (Richard Allan Scientific, Kalamazoo, MI). Sections wereexamined by polarized light microscopy. Photographs of six randomcortical fields (3400) from each animal were taken using the SPOTcamera and the percentage of positive tubulointerstitial area wasmeasured using the Optimas program.

TUNEL Assay of Apoptotic Nuclei. Apoptotic cells were de-tected in kidney sections using the terminal deoxynucleotidyl trans-ferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) as-say. To distinguish between proximal and distal tubules, weperformed Fx1A/TUNEL double immunostaining as described byHughes and Johnson (39). The Fx1A antibody that reacts with prox-imal tubular brush border antigens was a kind gift from Dr. WilliamCouser (University of Washington, Seattle, WA). Cells were regardedas TUNEL positive if their nuclei both were stained and had anapoptotic morphology characterized by typical nuclear pyknosis andchromatin condensation. The number of TUNEL-positive cells in eachspecimen was calculated in a blinded fashion by counting the numberof TUNEL-positive tubular and interstitial cells separately in anaverage of 12 sequentially selected nonoverlapping fields of renalcortex at3400 magnification. Results were expressed as the meannumber of apoptotic cells per3400 field.

Total Kidney Collagen ContentTotal renal collagen was measured biochemically as described

previously (40). In brief, an accurately weighed portion of the ob-structed kidney was homogenized in distilled water using a pelletpestle (Kontes Scientific Glassware/Instruments, Vineland, NJ). Ho-mogenates were hydrolyzed in 10 N HCl by incubation at 110°C for18 h. The hydrolysate was dried by speed vacuum centrifugation over3 to 5 h and redissolved in buffer (25 g of citric acid, 6 ml of glacialacetic acid, 60 g of sodium acetate, 17 g of sodium hydroxide in 500ml [pH 6.0]). Total hydroxyproline in this hydrolysate was determinedaccording to the chemical method technique of Kivirikkoet al. (41).Total collagen in the tissue was calculated on the assumption thatcollagen contains 12.7% hydroxyproline by weight. Final results wereexpressed asmg/mg kidney weight.

Statistical AnalysesAll values are expressed as mean6 1 SD, unless otherwise stated.

Results were analyzed by the Mann WhitneyU test using the SPSSprogram (SPSS, Inc. Cary, NC).P , 0.05 was considered statisticallysignificant.

ResultsKidney and Body Weights

The mean preoperative weights of the mice were similar inall experimental groups (Table 1). Most of the mice experi-

738 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 736–748, 2001

enced a small weight reduction in the postoperative period. Thewet weight of the experimental left kidney was significantlyincreased 3 d (1836 34 and 1866 27 for Timp-1andtimp-1,respectively) and decreased 14 d (936 10 and 1166 26) afterUUO operation compared with the wet kidney weight of thesham-operated kidneys (1386 18 and 1436 15). Fourteen dafter UUO operation, the mean kidney weight of theTimp-1group was less than thetimp-1 group, but the difference wasnot statistically significant (P 5 0.052).

Renal Expression of Genes Encoding Gelatinases andTIMP

Northern blot analysis of total kidney RNA from wild-typemice identified a faintTimp-1 mRNA band in the sham-operated mice and a significant increase inTimp-1 mRNAlevels 3 and 14 d after UUO operation (Figure 1). A very faintband was identified in thetimp-1mice consistent with previousreports and thought to represent an unstabletimp-1 mutanttranscript (20).

The possibility that deficiency of TIMP-1 resulted in com-pensation by increased expression of other protease inhibitorswas evaluated. Northern blot analysis using aTimp-2 cDNAprobe identified two transcripts of 3.5 kb and 1.0 kb. Comparedwith sham-operated kidneys, both transcripts were signifi-cantly increased 3 and 14 d after UUO operation with thesingle exception of the 3.5-kb transcript in the day 3 UUOtimp-1 group (Figure 2, A and B). In comparing the responseto UUO between theTimp-1and timp-1 groups, there was noconsistent difference. The 1.0-kbTimp-2transcript was signif-icantly higher in thetimp-1 group at 3 d, but it was signifi-cantly lower on day 14.

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

Compared with genetically identical sham groups, PAI-1mRNA levels were significantly increased in theTimp-1group

14 d after UUO operation and at both 3 and 14 d in thetimp-1group. At both 3 and 14 d, PAI-1 mRNA levels were signifi-cantly higher in thetimp-1 group compared with theTimp-1group. PAI-1 mRNA results for theTimp-1andtimp-1groups,respectively, expressed as fold increase relative to the lowestmean value were as follows: 3 d sham, 1.56 0.1versus1.060.4; 3 d UUO, 1.76 0.4 versus2.8 6 0.4; 14 d sham, 1.060.2 versus1.1 6 0.5; and 14 d UUO, 2.86 0.2 versus3.7 60.6.

Renal MMP-9 mRNA levels were significantly increased inboth UUO groups compared with the genetically identicalsham-operated control groups on days 3 and 14. No significantdifferences were observed betweenTimp-1 and timp-1 UUOgroups. Results for theTimp-1andtimp-1groups, respectively,expressed as fold increase relative to the lowest mean valuewere as follows: 3 d sham, 1.26 0.4 versus2.1 6 0.9; 3 dUUO, 3.66 1.2versus3.36 0.5; 14 d sham, 1.06 0.3versus1.5 6 0.2; and 14 d UUO, 3.76 1.4 versus3.4 6 0.6.

Hybridization using a MMP-2 cDNA probe identified twotranscripts of 4.0 kb and 3.2 kb. Renal mRNA levels of bothMMP-2 transcripts were significantly increased in the day 3and day 14 UUO groups compared with their sham-operatedcontrol groups. There was no significant differences in the4.0-kb transcript between theTimp-1and thetimp-1groups at3 and 14 d. Results for the 4.0-kb transcript in theTimp-1andtimp-1groups, respectively, expressed as fold increase relativeto the lowest mean value were as follows: 3 d sham, 1.16 0.3versus1.0 6 0.5; 3 d UUO, 2.66 0.6 versus2.8 6 0.6; 14 dsham, 1.56 0.5 versus1.0 6 0.7; and 14 d UUO, 8.16 1.5versus6.7 6 1.1. In contrast, at 14 d, the 3.2-kb transcriptmRNA levels were significantly higher in theTimp-1 groupcompared with thetimp-1 group (24.26 1.1 versus20.2 61.3-fold increase).

Renal Metalloproteinase ActivityGelatin zymography detected strong lytic bands representing

MMP-9 activity and considerably smaller bands due to MMP-2activity. After UUO operation, renal MMP-9 activity wasdecreased at 14 d in theTimp-1group and at 3 and 14 d in thetimp-1 group compared with their sham control groups (P 50.05; n 5 3 per group; Figure 3). However, basal MMP-9

Table 1. Body weight

Group n Before Operation(g)

Before Killing(g) Delta (%)

Timp-1 3 d UUO 8 266 3 236 3 13Timp-1 3 d sham 8 256 1 236 2 9timp-1 3 d UUO 8 256 3 216 3 15timp-1 3 d sham 8 256 2 226 2 13Timp-1 14 d UUO 8 266 3 236 3 12Timp-1 14 d sham 8 246 2 226 2 8timp-1 14 d UUO 8 246 3 236 2 6timp-1 14 d sham 8 256 2 256 2 3

Results are mean6 1 SD.


activity in the sham kidneys was higher in thetimp-1kidneys,and 3 and 14 d after UUO operation, mean MMP-9 activitywas higher in thetimp-1 group compared with theTimp-1group (P 5 0.05). In contrast, UUO induced MMP-2 activity,and there was no difference between theTimp-1and thetimp-1groups. Clear bands of renal caseinolytic activity were notdetected in any of the experimental groups (data not shown).

MMP-9 Western BlottingTo investigate further the discrepancy between increased

MMP-9 mRNA levels and decreased MMP-9 gelatinase activ-ity, we evaluated MMP-9 protein levels by Western blotting.The findings were consistent with the results of the gelatinzymography showing decreased MMP-9 protein in the UUOgroups compared with the sham groups (Figure 4).

TIMP-1 Protein and ActivityWestern blotting and reverse zymography were unsuccessful

in detecting convincing bands corresponding to TIMP-1 pro-

tein and inhibitory gelatinolytic activity, respectively, withinkidney extracts (data not shown).

Severity of Renal FibrosisTotal kidney collagen was significantly increased 14 d after

UUO compared with sham-operated kidneys, but there was nodifference found between theTimp-1 and thetimp-1 groups(Figure 5). Significant interstitial fibrosis was confirmed his-tologically by immunofluorescence staining for collagen III(Figure 6) with no significant differences between theTimp-1and thetimp-1 groups. Similar findings were observed whenthe severity of fibrosis was evaluated by picrosirius red stain-ing. Results for theTimp-1 and timp-1 groups, respectively,expressed as percentage of tubulointerstitial area stained wereas follows: 3 d sham, 0.26 0.2 versus0.3 6 0.1; 3 d UUO,0.56 0.4versus0.66 0.3; 14 d sham, 0.26 0.1versus0.560.3; and 14 d UUO, 4.46 1.1 versus4.2 6 1.8.

Figure 1.Timp-1 Northern blot autoradiograph (A) and graph of mean band density (B). Results are expressed as mean fold increase6 1 SDwith the baseline value defined as the density of the weakest band on the Northern blot. The 28-s band from the ethidium bromide–stained gelis 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 , 0.05versusday 3Timp-1UUO group; ***, P , 0.05versusday 14Timp-1UUO group.


TGF-b and Matrix Protein Gene ExpressionNorthern blot analysis confirmed that there was no differ-

ence betweenTimp-1 and timp-1 mice with respect to theinduction of genes encoding TGF-b and the matrix genesa1(III) procollagen and fibronectin in response to UUO. Com-pared with sham-operated kidney, renal mRNA levels for thesegenes were significantly increased 3 d (procollagen III, 26.364.5versus26.36 3.6; fibronectin, 23366 907versus24886873; TGF-b, 5.0 6 0.6 versus5.5 6 0.7-fold increase abovelowest mean sham group value) and 14 d (procollagen III,29.66 6.5versus29.06 3.0; fibronectin, 33416 1586versus

32816 980; TGF-b, 5.86 1.2versus5.86 0.7-fold increase)after UUO operation.

Interstitial Myofibroblasts, Macrophages, and TubularApoptotic Cells

The number ofa-smooth muscle actin–positive interstitialmyofibroblasts was significantly increased 3 and 14 d afterUUO operation and was similar in intensity between theTimp-1and timp-1 groups (Figure 7). A significant interstitialinfiltrate of F4/80-positive interstitial macrophages developedin response to UUO and was similar in theTimp-1andtimp-1

Figure 2. Northern blot analysis of renal protease inhibitorsTimp-2 and Timp-3. (A and C) autoradiographs of the Northern blot with theethidium bromide–stained 28-s ribosomal band illustrated below each lane. (B and D) Results of analysis of the band density expressed as meanfold increase6 1 SD with the baseline value defined as the density of the weakest band on the Northern blot.Timp-1, wild-type mice expressingthe normal allele;timp-1, TIMP-1–deficient mice expressing the mutant allele. *,P , 0.05 compared with sham group of same genotype; **,P , 0.05versusday 3Timp-1UUO group; ***, P , 0.05versusday 14Timp-1UUO group.


groups at day 14 (Figure 8). This interstitial infiltrate appearedmore rapidly (day 3) in thetimp-1 group but was similar inintensity to theTimp-1UUO group on day 14. The numbers of

TUNEL-positive interstitial and proximal tubule cells weresignificantly increased 3 and 14 d after UUO operation (Figure9). The number of apoptotic interstitial cells was significantlyhigher in thetimp-1 UUO groups compared with theTimp-1UUO groups. There was no difference in the number of apo-ptotic proximal tubule cells. Results for theTimp-1andtimp-1groups, respectively, expressed as the number of TUNEL-positive tubular cells per3400 field were as follows: 3 d sham,0 6 0 versus0 6 0; 3 d UUO, 0.076 0.08versus0.096 0.08;14 d sham, 06 0 versus0.026 0.04; and 14 d UUO, 0.360.2 versus0.5 6 0.2. Very few TUNEL-positive distal tubulecells were observed after UUO (data not shown).

DiscussionThis study detected a striking 20- to 40-fold increase in renal

Timp-1mRNA levels in mice in response to ureteral obstruc-tion, a finding that has been reported previously in rats (13) andrabbits (12). In fact, upregulated expression ofTimp-1mRNAhas been observed as an almost universal feature of solid organfibrosis, be it in the kidney (6), lung (42), or liver (43). Atemporal association has been made between normalization ofTimp-1mRNA levels and reversal of hepatic fibrosis in a ratmodel of CCl4-induced liver fibrosis, suggesting a relationshipbetween TIMP-1–dependent inhibition of collagen turnoverand liver fibrosis (43). Despite the numerous associations

Figure 3. Gelatin gel zymography illustrating renal MMP-9 andMMP-2 activity (A). The density of the lytic bands expressed as meanarbitrary units6 1 SD is summarized in B.Timp-1, wild-type miceexpressing the normal allele;timp-1, TIMP-1–deficient mice express-ing the mutant allele. *,P 5 0.05 compared with sham group of samegenotype (n 5 3 per group); **,P 5 0.05versusday 3Timp-1UUOgroup (n 5 3 per group); ***, P 5 0.05versusday 14Timp-1UUOgroup (n 5 3 per group).

Figure 4. MMP-9 Western blot of kidney extracts. Each lane wasloaded with 20mg of total kidney protein.Timp-1, wild-type miceexpressing the normal allele;timp-1, TIMP-1–deficient mice express-ing the mutant allele.

Figure 5. Total kidney collagen. Results are meanmg collagen/mgkidney wet weight6 1 SD. Timp-1, wild-type mice expressing thenormal allele;timp-1, TIMP-1–deficient mice expressing the mutantallele. *, P , 0.05 compared with sham group of same genotype.


between TIMP-1 and fibrotic states, definitive proof thatTIMP-1 plays a significant pathogenetic role is still lacking.The results of the present study demonstrate clearly that ge-netic deficiency of TIMP-1 fails to attenuate the severity ofrenal fibrosis after UUO. These results are consistent with ourrecent findings in a much less aggressive model of renalinterstitial fibrosis induced by protein-overload proteinuria(17).

The most likely explanation for these findings is geneticredundancy with compensation for a TIMP-1–deficient stateby another MMP inhibitor. Likely candidates include TIMP-2and TIMP-3. TIMP-2 is constitutively expressed in the kidney.In response to UUO, 2- to 14-fold increases inTimp-2mRNA

were observed. Although there was no difference in theTimp-2response between the TIMP-1-deficient and wild-type mice, itis possible that this inhibitor was present in sufficient quantityto 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. How-ever, in comparing the response in the TIMP-1–deficient andwild-type mice,Timp-3mRNA levels were significantly higherat both 3 and 14 d after UUO operation in the TIMP-1–deficient mice. TIMP-3 is unique among the known MMPinhibitors because of its affinity for and exclusive localizationwithin extracellular matrix (16). It may be especially effectiveas an MMP inhibitor at extracellular sites of active fibrosis.

Figure 6. (A) Photomicrographs of interstitial collagen III deposition detected by indirect immunofluorescence staining. The percentage oftubulointerstitial area stained expressed as mean6 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,3400.


Changes in PAI-1 expression were unexpected and deservefurther investigation. As reported in several studies, inductionof PAI-1 gene expression is frequently observed in fibroticdisease states (6). This serine protease inhibitor is thought tohave profibrotic effects as a consequence of decreased gener-ation of plasmin. Plasmin is known to be a potent activator ofprometalloproteinasesin vitro, and a similar rolein vivo hasbeen proposed (44). Renal PAI-1 mRNA levels were signifi-cantly higher in the TIMP-1- deficient mice at both 3 and 14 dafter UUO operation. We recently found that the severity ofrenal fibrosis is significantly reduced in PAI-1–deficient micecompared with wild-type mice after UUO operation (Odaet al., manuscript submitted), raising the distinct possibilitythat these differences in PAI-1 expression are relevant to thefindings in the present study.

An important question is whether the differences in TIMP-1expression were associated with functional differences in renalMMP activity. This study focused on gelatinolytic activity fortwo reasons. First, gelatinase activity is the strongest MMPactivity currently recognized in the kidney. In fact, when thegel substrate was changed from gelatin to casein in the presentstudy, convincing lytic bands were barely visible. Second,TIMP-1 has high specificity for MMP-9, and it also binds topro-MMP-9 (16). In addition to gelatin, MMP-9 (92-kD typeIV collagenase or gelatinase B) also degrades collagens typeIV and V, fibronectin, laminin, entactin, elastin, and proteo-glycans; 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 ex-

Figure 7. (A) Photomicrographs of interstitial myofibroblasts detected by immunohistochemical staining fora-smooth muscle actin. Thepercentage of tubulointerstitial area stained expressed as mean6 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,3400.


pressed in the kidney and inhibited by TIMP-1. Of particularnote is that gelatinases do not degrade fibrillar collagens suchas collagen type I and III that also accumulate in the renalinterstitium during fibrosis. Also unknown is whether each ofthe matrix proteins that constitutes the interstitial scar isequally detrimental to renal structure and function. Once thesequestions are resolved, we will be better positioned to focus onthe specific proteases and protease inhibitors that regulate theturnover of the critical constituents of the interstitial scar.

In the present study, a strong gelatinolytic band correspond-ing to MMP-9 activity was present in extracts prepared fromsham kidney homogenates. This activity was significantly de-creased after UUO operation. However, baseline MMP-9 ac-tivity was significantly higher in the TIMP-1–deficient miceand remained significantly higher than levels observed in thewild-type mice 3 and 14 d after UUO operation. An interestingfinding was a significant increase in renal MMP-9 mRNAlevels after UUO operation. Because the expression of MMP is

Figure 8.(A) Photomicrographs of interstitial monocytes/macrophages detected by indirect immunofluorescence staining for the F4/80 antigen.The number of positive cells per3400 field expressed as mean6 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 , 0.05versusday 3Timp-1UUO group. Magnification,3400.


controlled primarily at the level of transcription (47), thisdifference between MMP-9 mRNA and activity suggests eitherdecreased activation of MMP-9 or inhibitor-dependent inacti-vation of MMP-9 activity. In data not shown, pretreatment ofkidney extracts with 4-aminophenylmercuric acetate to activatelatent MMP-9 did not result in a significant increase in MMP-9activity. Furthermore, Western blotting identified a single dis-tinct band. These findings argue against the decreased activa-tion hypothesis and favor the conclusion that the MMP-9activity was decreased by its inhibitors.

Renal MMP-2 activity is considerably lower than MMP-9activity in normal kidneys. In response to UUO, both MMP-2mRNA and activity levels were significantly increased. Thedegree of change was similar in the TIMP-1–deficient andwild-type mice with the single exception that the less abundant3.2-kb transcript was significantly higher in the TIMP-1–de-ficient group on day 14.

Several other key aspects of the interstitial fibrogenic re-sponse were evaluated and found to be similar between the

Timp-1 and timp-1 groups, indicating that the TIMP-1 defi-ciency did not induce other compensatory responses. In par-ticular, the expression of TGF-b and matrix genesa1(III)procollagen and fibronectin were similar. Infiltration of mono-cytes into the interstitium occurred more rapidly (day 3) in theTIMP-1–deficient group, but this difference was no longerevident on day 14. It is noteworthy that a unique feature oftubulointerstitial disease observed in our studies with PAI-1–deficient mice was a significant reduction in the number ofinterstitial macrophages (Odaet al., manuscript submitted).Whether the increased expression of PAI-1 in the TIMP-1–deficient mice facilitated early macrophage recruitment isspeculative.

The observation that the severity of renal fibrosis was un-affected by the absence of TIMP-1 raises the possibility thatTIMP-1 may be mediating other effects in the kidney. Themultifunctionality of TIMP-1 is increasingly recognized (16).What this effect might be remains unknown. TIMP-1 has beenreported to inhibit B-cell death by apoptosis (48). The results

Figure 9.TUNEL-positive tubulointerstitial cells. Photomicrographs illustrate (arrows) a positive interstitial cell (A) and a positive proximaltubule cell (B) in a mouse with UUO. Proximal tubular cells are indicated by the dark brush border staining reaction with the anti-Fx1Aantibody. The number of TUNEL-positive interstitial cells (C) is expressed as mean number of positive cells per3400 field 6 1 SD.Magnification,3630 in A and B.


of the present study suggest that TIMP-1 may play a similarrole in the kidney as the deficiency of TIMP-1 resulted in asignificant increase in the number of apoptotic interstitial cells3 and 14 d after UUO operation. The functional significance ofthis difference is unclear. TIMP-1 has also been reported toinhibit smooth muscle cell migration (49). Although the originof the interstitial myofibroblasts that typify progressive renaldisease remains controversial, in the present study the numberof interstitial a-smooth muscle actin–positive interstitial cellswas not altered in the TIMP-1–deficient mice with UUO,suggesting that TIMP-1–dependent inhibition of perivascularcell migration into the interstitium is not a feature of renalfibrosis.

A major limitation of the present study was the inability tolocalize TIMP-1 protein and to demonstrate TIMP-1 activity inthe kidney. Similar difficulties were encountered in other ex-perimental systems as well. The anti–TIMP-1 antibody used inthese studies is reported to react with mouse TIMP-1, but it didnot produce a staining pattern or a reaction on Western blotsthat was convincingly different from background results pro-duced when the secondary antibody was used alone despitemany different blocking procedures. Results from our previousstudies in rats identified both tubular and interstitial cells as theintrarenal source of TIMP-1 production after injury, and wepredict that the same would be true in mice (50). Reversezymography to detect MMP inhibitor activity has proved tech-nically challenging when efforts are made to isolate the inhib-itors from tissue rather than cultured cells or their supernatant.The procedure used in the present study produced clear bandsof activity when recombinant TIMP-1, -2, or -3 were used, butthe only convincing activity observed in the kidney extractscorresponded to TIMP-3 activity (data not shown).

In summary, the fibrogenic response to UUO in mice in-cludes a significant upregulation ofTimp-1. However, despitegenetic deficiency of TIMP-1 and higher renal MMP-9 activ-ity, the severity of renal interstitial fibrosis was not reduced inthe TIMP-1–deficient mice. Significant differences in the renalexpression of TIMP-3 and PAI-1 and/or high constitutive ex-pression of TIMP-2 may compensate when TIMP-1 is notavailable during the active phase of renal fibrosis. Futurestudies in mice that are genetically deficient in TIMP-2 andTIMP-3 and in mice that have combined deficiencies inTIMP-1 and PAI-1, TIMP-2, and/or TIMP-3 should be able toclarify the mechanism of compensation and establish the roleof impaired matrix turnover in the renal fibrogenic response.However, an alternative explanation is that inhibition of intrin-sic MMP activity does not represent a profibrotic event in thekidney.

AcknowledgmentsThis work was funded by National Institutes of Health Grant No.

DK54500 (A.A.E.). The authors acknowledge the outstanding tech-nical assistance of Dr. Richard D. Kenagy with zymographic studiesand Dr. Jeremy Hughes with the TUNEL assay. The assistance ofRuthanne Naranjo with reference formatting is greatly appreciated.

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timp-1 deficiency does not attenuate interstitial fibrosis in obstructive nephropathy

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