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Experimental and Toxicologic Pathology 59 (2007) 1–7

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Expression and significance of a disintegrin and metalloproteinase with

thrombospondin motifs (ADAMTS)-1 in an animal model of renal

interstitial fibrosis induced by unilateral ureteral obstruction

Atsushi Nakamura�, Yukiko Sakai, Chieri Ohata, Toshi Komurasaki

Medicinal Research Laboratories, Taisho Pharmaceutical Co., LTD, 1-403 Yoshino-Cho, Kita-Ku, Saitama-shi 331-9530,

Saitama, Japan

Received 1 November 2006; accepted 11 January 2007

Abstract

To evaluate the pathological roles of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 in rat renal interstitial fibrosis, we examined the expression, localization and effect on growth of ADAMTS-1 in anormal rat kidney cell line (NRK-49F). Increased ADAMTS-1 mRNA expression was observed in the kidney by insitu hybridization after induction of unilateral ureteral obstruction (UUO) in male Wistar rats, the mRNA waslocalized in the renal tubular epithelial cells in the outer stripe of the outer medulla in the UUO kidney. On the otherhand, no positive signals were observed in the sham-operated-kidney. Western-blot analysis of stable humanembryonic kidney 293 (HEK293) transformant cells expressing rat ADAMTS-1 containing the V5 tag using anti-V5tag monoclonal antibody revealed the presence of two post-translationally processed bands in the cells: an 87-kDaband with a metalloproteinase motif and 65-kDa band with a thrombospondin motifs. On the other hand, secretion ofthe 65-kDa protein into the culture supernatants from the transformant cells was confirmed. Treatment with theculture supernatant of the transformant cells potently reduced the uptake of 3H-thymidine in the NRK-49F cells, nosuch inhibitory effect was observed with the culture medium of the HEK293 cells. These results suggest that the UUO-induced expression of ADAMTS-1 in the rat renal tubular epithelial cells may actively contribute to the inhibition ofDNA synthesis in the renal interstitial fibroblasts via the 65-kDa moiety with thrombospondin motifs.r 2007 Elsevier GmbH. All rights reserved.

Keywords: ADAMTS-1; Renal interstitial fibrosis; Unilateral ureteral obstruction; In situ hybridization; HEK293; NRK-49F;

Thymidine uptake

Introduction

A cellular disintegrin and metalloproteinase withthrombospondin motifs (ADAMTS) represent a pro-tease family found both in mammals and invertebrates,and have been shown to play important roles in

e front matter r 2007 Elsevier GmbH. All rights reserved.

p.2007.01.003

ing author. Fax: +8148 652 7254.

ess: a.nakamura@po.rd.taisho.co.jp (A. Nakamura).

proteolytic modification of cell-surface proteins andthe extracellular matrix (Porter et al., 2005). ADAMTS-1 was first cloned from colon adenocarcinoma cells, andis distinguished from other ADAM molecules by thepresence of thrombospondin type I motifs at itsC-terminal (Kuno et al., 1997; Kuno and Matsushima,1998). Investigation of the biological role of ADAMTS-1 in the kidney using ADAMTS-1-null mutant micerevealed that it is required for normal morphological

ARTICLE IN PRESSA. Nakamura et al. / Experimental and Toxicologic Pathology 59 (2007) 1–72

development and function of the kidney (Yokoyamaet al., 2002; Mittaz et al., 2005).

Renal interstitial fibrosis is known as the finalcommon pathway to end-stage renal failure (Nath,1992; Nangaku, 2004). Abnormal accumulation ofextracelluar matrix (ECM) components, in particular,of collagen type III, in the tubular interstitial area, leadsto impairment of renal function (Teppo et al., 2003).The regulation of collagen mass is reflected by thedynamic balance between its synthesis and degradation(Laurent, 1987). The collagen turnover is regulated bythe expression of metalloproteinases (MMPs) and theirendogenous inhibitors, the tissue inhibitors of metallo-proteinases (TIMPs) (Eddy, 1996; Norman and Fine,1999). The genes encoding these proteins expressed inthe early stages of the proximal tubular degeneration-interstitial fibrosis sequence are likely to play animportant role in the renal failure.

Multiple functions of ADAMTS-1, including boneturnover and formation (Miles et al., 2000; Lind et al.,2005), angio-inhibitory activity (Vazquez et al., 1999),uterine tissue remodeling (Kim et al., 2005) andatherogenesis promotion (Jonsson-Rylander et al.,2005) have been proposed. While ADAMTS-1 expres-sion has been reported in the articular chondrocytes ofboth normal and arthritic mice, no abrogation ofaggrecan loss has been demonstrated in antigen-inducedarthritis in ADAMTS-1-null mice (Little et al., 2005).The presence of the disintegrin/cystein-rich domain inhuman ADAMTS-1, in which Glu385 may be thecatalytic residue, has been shown to be essential for itsenzymatic activities (Wei et al., 2002). HumanADAMTS-1 is processed into two post-translationalproducts, namely, p87 and p65 (Rodriguez-Manzanequeet al., 2000), and p65 has been suggested to have apossible role in the proximal tubular degeneration-interstitial fibrosis relationship.

Materials and methods

Induction of renal interstitial fibrosis in rats

Male Wistar rats were obtained from Charles RiverJapan (Yokohama, Japan) and acclimated until use. Theanimals were maintained under controlled conditions,including a 12 h light/12 h dark cycle, constant tempera-ture of 2373 1C, and relative humidity of 50720%.Animal care and use conformed to the guidelinesestablished by the Institutional Animal Care and UseCommittee of Taisho Pharmaceutical Company, Ltd.Thirty-six rats, 8 weeks of age, were used. Unilateralureteral obstruction (UUO) was executed in the animalsunder Nembutal (Dainippon Sumitomo Pharma,Osaka, Japan) anesthesia by ligation of the left ureter

with sterilized 5-0 silk Matsuda sutures (Tokyo, Japan).Sham-operated animals were used as controls. The ratswere then sacrificed by exsanguination under anesthesiaon Day 3 after the operation, and the left kidneys wereused for the following studies.

In situ hybridization (ISH)

For histological examination, the left kidney wasremoved and longitudinally divided; one half wasimmediately fixed in neutralized 10% formalin buffersolution (Wako, Osaka, Japan) for 2 or 3 days and thenembedded in paraffin. The technique of ISH on paraffinsections using digoxigenin (DIG)-labeled RNA probeshas been reported previously (Ishii et al., 2004). For thepreparation of the RNA probes of rat ADAMTS-1(NIH accession ]NM_024400), a 201-bp sequence wasamplified by PCR using forward and reverse primersand EX Taq polymerase (Takara, Ohtsu, Japan) (primersequences: forward, 50-CATGGCTGACTTCCACGG-CA-30, and reverse, 50-GCCAGCTACAGAAATTCC-TAAGGG-30), and the PCR product was ligated intothe pTBlue vector (Novagen, Madison, WI, USA). TheT7 promoter containing the ADAMTS-1 PCR productwas PCR-amplified with forward or reverse primers andthe T7 promoter adaptor primer. Dig-labeled RNAprobes were synthesized by in vitro transcription usingthe T7 Polymerase Transcription System (Roche Diag-nostics, Indianapolis, IN, USA).

cDNA cloning and ADAMTS-1 expression in

HEK293 cells

ADAMTS-1 cDNA was cloned from a rat UUOkidney by RT-PCR using KOD polymerase (Toyobo,Tokyo, Japan). The cloned cDNA was inserted intothe TOPO site of pcDNA3.1 (Invitrogen, Tokyo, Japan)to produce an expression vector for ADAMTS-1/pcDNA3.1. HEK293 cells were grown in Dulbecco’sModified Eagle Medium (D-MEM, Invitrogen) supple-mented with 10% (v/v) fetal bovine serum (FBS,Invitogen) in a 5% CO2 atmosphere at 37 1C. TheHEK293 cells were then transfected with the ADAMTS-1/pcDNA3.1 expression vector using Magnetofec-tionTM (OZ Bioscience, Marseille, France) in accor-dance with the manufacturer’s instructions. For theexpression analysis of ADAMTS-1, the stable transfor-mant cells were maintained in D-MEM supplementedwith 1% (v/v) FBS and 100 mg/ml of G418 (Invitrogen)in a CO2 incubator.

Immunocytochemistry

After the transformant cells cultured on SlideChamber (BD Biosciences, Tokyo, Japan) were fixed

ARTICLE IN PRESSA. Nakamura et al. / Experimental and Toxicologic Pathology 59 (2007) 1–7 3

with ice-cold acetone for 30min, they were washed with10mM phosphate-buffered saline (PBS, pH 7.4) andincubated with a monoclonal antibody directed againstV5 (Invitrogen, diluted 1/2000). The immunocytochem-ical detection was performed in a Ventana NX Auto-mated Immunohistochemistry System (Ventana Japan)in accordance with the manufacturer’s instructions.

Western blotting

Extracts of the ADAMTS-1 transformant cells wereprepared for western blotting using the M-PERR

Mammalian Protein Extraction Reagent (Pierce, Rock-ford, IL, USA) containing CompleteTM Proteinaseinhibitor cocktail tablets (Roche Diagnostics), in accor-dance with the manufacturer’s instructions. After SDS-PAGE carried out according to the standard method on10% gels (Laemmli, 1970), the proteins were transferredon to Immuno-BlotTM PVDF Membrane for ProteinBlotting (Nippon Bio-Rad Laboratories, Tokyo Japan).The membrane was blocked for 1 h with BlockingReagent (Roche Diagnostics) and rinsed three timesfor 5min each in TTBS washing buffer (pH 7.4)containing 20mM Tris, 500mM NaCl and 0.1% Tween20. Detection of ADAMTS-1 was performed byincubation with an alkaline phosphatase-conjugatedantibody directed against the V5 tag (Invitrogen, mousemonoclonal IgG; diluted 1/2000) for 1 h at roomtemperature. The blots were developed with Lumi-Imager F1TM (Roche Diagnostics) detection of thechemiluminescense was performed with Immun-StarTM

AP Substrate (Bio-Rad), in accordance with themanufacturer’s instructions.

Gelatin zymography

Gelatinase activity was determined using a modifica-tion of the method reported in the literature (Sasakiet al., 2005). In brief, a 10% polyacrylamide gel of0.75-mm thickness containing 0.1mg/ml gelatin wasused and the proteins were separated using Laemmli’sbuffer system. Extracts of the transformant andHEK293 cells for measurement of the gelatinase activitywere prepared using M-PERR without the proteinaseinhibitor. As molecular weight markers for the gelatinzymography (Life laboratory Co., Yamagata, Japan),three recombinant matrix metalloproteinases (MMPs),namely, proMMP-9 (p92), proMMP-2 (p72) and MMP-2 (p62) were used. An aliquot of the cell extracts wasmixed with an equal amount of Laemmli’s sample buffer(Bio-Rad) and subjected to electrophoresis withoutheating. The gel was then rinsed with 2.5% TritonX-100 for 1 h at room temperature and incubated in thereaction buffer (10mM CaCl2, 50mM Tris-HCl, pH 7.4,and 0.02% NaN3) for 16 h at 37 1C, fixed with 50%

methanol–10% acetic acid for 30min, stained withPAGE Blue 83 (Daiichi Pure Chemicals, Tokyo, Japan)for 1 h, and destained with 20% methanol–10% aceticacid until clear white bands appeared against thebackground.

NRK-49F culture and uptake of 3H-thymidine

The NRK-49F cell line derived from normal ratkidney was obtained from Dainippon-pharma (Osaka,Japan), with the adherent fibroblast phenotype andexpressing the epidermal growth factor receptor (deLarco and Todaro, 1978). DNA synthesis in the NRK-49F cells was assessed by measuring [3H]-thymidineincorporation. Briefly, the cells were seeded at a densityof 1� 105 cells/well in 24-well culture plates (Nunc,Tokyo, Japan). After being cultured in D-MEMcontaining 1% FBS for 18 h, the NRK-49F cells weretreated with the conditioned medium of ADAMTS-1transformant or HEK cells, followed immediately by theaddition of 1 mCi/ml/well of [3H]-thymidine (AmericanRadiolabeled Chemicals, St. Louis, USA). After incuba-tion for 2 h at 37 1C, the medium was aspirated, and thecells were washed three times with ice-cold PBS, andthen incubated with ice-cold 5% trichloroacetic acid(TCA, Wako) for 15min. After aspiration of the TCA,the cells were dissolved in lysis buffer containing 0.4NNaOH, and an aliquot was analyzed by liquid scintilla-tion counting (LS6500, Beckman Coulter, Fullerton,CA, USA). The results were expressed as the mean andstandard deviation of the determination from 12replicates of each sample. The statistical differenceswere determined with Student’s t-test using the Excelsoftware (Microsoft Office).

Results

Localization of ADAMTS-1 mRNA by ISH

UUO induced renal interstitial fibrosis in the outerstripe of the outer medulla, characterized histologicallyby dilatation of the renal tubules and excessive growthof interstitial fibroblasts. The pathological severity ofthe renal interstitial fibrosis was markedly aggravated byDay 3 after the UUO. To examine the expression andlocalization of the ADAMTS-1 mRNA in the UUOkidney, we performed ISH on paraffin sections of thekidney on Day 3 after the execution of the UUO.Whereas intense blue signals were detected with theantisense RNA probe of ADAMTS-1 in the dilatedtubular epithelial cells of the UUO kidney, no signalswere detected with the sense RNA probe. In the sham-operated kidney, no positive signals were detected witheither the antisense or the sense RNA probe (Fig. 1).

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Fig. 1. In situ hybrization for rat ADAMTS-1 in the kidney: (a, c) UUO kidney and (b, d) sham-operated kidney. Intense blue

signals representing ADAMTS-1 expression were seen with the antisense RNA probe in the dilated tubular epithelial cells of the

UUO kidney (a), no signals are seen with the antisense RNA probe in the sham-operated kidney (b), no signals were observed in

either the UUO or the sham kidney with the sense RNA probe (c, d). Original magnification 200� .

Fig. 2. Characterization of transformant cells expressing rat ADAMTS-1 or HEK293 cells: (a) RNA expression in the transformant

cells by RT-PCR, (b) immunocytochemical detection of ADAMTS-1 expressed in the transformant cells using an anti-V5 tag

monoclonal antibody (c) Western-blot analysis for the ADMATS-1 protein in the extract of the transformant cells and culture

supernatant of the transformant cells. Note a single band of p65 protein was seen in the culture supernatant of the transformant

cells.

A. Nakamura et al. / Experimental and Toxicologic Pathology 59 (2007) 1–74

ARTICLE IN PRESSA. Nakamura et al. / Experimental and Toxicologic Pathology 59 (2007) 1–7 5

Analysis of rat ADAMTS-1 expression in a stable

transformant cell line

The expression vector of rat full-length cDNA clonedfrom the UUO kidney under the control of a cytome-galovirus promoter was transfected into HEK293cells. RT-PCR analysis to analyze the expression ofADAMTS-1 at the mRNA level in the stable transfor-mant or HEK293 cells revealed a single bandof ADAMTS-1 in the transformant cells, but notHEK293 cells (Fig. 2a). Western blotting using anti-V5tag monoclonal antibody also confirmed the detection ofthe ADAMTS-1 protein with two bands, p87 and p65,in the extract of the transformant cells. ADAMTS-1 waslocalized throughout the cytoplasm as an intense brownsignal in the transformant cells (Fig. 2b). Furthermore,the transformant cells were cultured to confluence for3 days in culture medium containing 1% FBS, andwestern blotting revealed a single band of the p65protein, in the culture supernatant of the transformantcells (Fig. 2c).

Detection of the gelatinase activity of ADAMTS-1

by gelatin zymography

The gelatinase activity of the transformant cellsexpressing ADAMTS-1 was detected by gelatin zymo-graphy. Although the gelatinase activities of recombi-nant proMMP-9 (p92), proMMP-2 (p72) and MMP-2(p62) were detected as clear bands, no gelatinase activitywas found in the extracts of either the tranformant cellsor the HEK293 cells (Fig. 3).

Effect of the culture supernatant of the transformant

cell on the 3H-thymidine uptake by the NRK-49F

cells

To determine if the p65 protein secreted into theculture supernatant of the transformant cells had apossible effect on the regulation of cell growth of theNRK-49F cells, the 3H-thymidine uptake experimentwas performed. Comparison of the effects of treatmentof the NRK-49F cells with the culture supernatant ofthe transformant cells and HEK293 cells revealed thatthe uptake of 3H-thymidine was decreased by 66%following treatment with the p65 protein-containingculture supernatant of the transformant cells as com-pared to that following treatment with the culturesupernatant of the HEK293 cells (Fig. 4). The statisticalsignificance of the difference in the inhibitory effecton the 3H-thymidine uptake between the two cul-ture supernatants was confirmed by Student’s t-test(po0.001).

Discussion

Confirmation of the upregulation of ADAMTS-1 inthe rat UUO kidney in this study may suggest theexistence of a relationship between the MMP activity ofADAMTS-1 and the pathogenesis of renal interstitialfibrosis. Although the gelatinase activity of ADAMTS-1was confirmed in an insect expression system, only weakor highly unstable enzyme activity of gelatinase wasfound in eukaryotic cells (Kuno et al., 2000; Rodriguez-Manzaneque et al., 2002; Lind et al., 2006). Therefore,we attempted to determine the gelatinase activity byconducting gelatin zymography using recombinantADAMTS-1 expressed in HEK293 cells; however, nogelatinase activity was observed in the extract of thetransformant cells despite detection of the active ofrecombinant MMPs. Furthermore, versican, a substrateof ADAMTS-1, was localized in the collecting tubules inboth the UUO and sham-operated kidneys, with noexpression in the proximal and distal tubules or theinterstitial area (unpublished data), while ADAMTS-1expression was limited to the proximal or distal tubules.The transformant cell experiments showed that the post-translational product of the p87 protein with ametalloproteinase motif of ADAMTS-1 was a non-secreted phenotype. These results suggest that there islittle possibility of the p87 protein participating in theaggravation of interstitial fibrosis in the rat UUOkidney.

Also, the transformant cell experiments showed notonly intracellular post-translational processing ofADAMTS-1 to p87 and p65, but also secretion of p65into the culture supernatant following stable expressionof ADAMTS-1 in HEK293 cells. Overexpression of onlythe C-terminal half region of mouse ADAMTS-1,representing the p65 protein, was reported to suppressChinese hamster ovary (CHO) tumor growth in mice(Kuno et al., 2004). Therefore, the p65 protein may havea potential physiological role in cell growth. In thisstudy, since the inhibitory effects of the p65 protein onthe cell growth of NRK-49F cells was demonstrated by3H-thymidine uptake experiments, the p65 protein ofADAMTS-1 might have an inhibitory effect on the cellgrowth of normal kidney fibroblasts. This is the firstreported evidence of the possible role of the p65 proteinof ADAMTS-1 in cell growth inhibition during thepathogenesis of fibrosis.

Since the p65 protein appears to be immediatelysecreted into the extracellular space, interaction of thep65 protein with its receptor may be a possiblemechanism of its inhibitory effect on cell growth.However, the existence of a receptor for the p65 proteinof ADAMTS-1 has not yet been demonstrated. The p65protein has two thrombospondin-1 (TSP-1) motifs in itsamino acid sequence, and CD36 and CD47 have beenreported as the binding proteins for these TSP-1 motifs.

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Fig. 3. Detection of gelatinase in the cell extract of transfor-

mant or HEK293 cells by zymography. Gelatinase activity was

seen in the MMPs sample including proMMP-9 (p92),

proMMP-2 (p72), MMP-2 (p65) as a clear bands. No

gelatinase band was seen in the extract of transformant or

HEK293 cells.

Fig. 4. Effects of 3H-thymidine uptake on NRK-49F cells

treated with culture supernatant of transformant or HEK293

cells. Inhibition with 34% was seen in NRK-49F treated with

transformant supernatant compared to HEK293 supernatant,

statistically significant differences between transformant and

HEK293, n ¼ 12, po0.001.

A. Nakamura et al. / Experimental and Toxicologic Pathology 59 (2007) 1–76

CD36 is a member of the scavenger receptor type Bfamily implicated in the binding of lipoproteins,phosphatidylserine, TSP-1, and the uptake of long-

chain fatty acids (Pearce et al., 1998). As CD47 is knownto be an integrin-associated protein, a possible role ofthis protein in inhibiting taxane-induced toxicity hasbeen reported (Lih et al., 2006). Anyway, further studiesto elucidate the roles of CD36 and CD47 in renalinterstitial fibrosis are needed to clarify the functions ofADAMTS-1.

In this study, we clarified the de novo expression ofADAMTS-1 in the renal tubular epithelial cells of thekidney after UUO, and the p65 protein secreted afterpost-translational processing of ADAMTS-1 was foundto inhibit the cell growth of normal rat fibroblast cells.We postulate that the C-terminal p65 protein ofADAMTS-1 has therapeutic potential as an inhibitorof fibroblast growth against interstitial fibrosis resultingfrom renal injury.

Acknowledgments

We thank Miss Hanako Togashi and Yuki Iijima fortechnical assistance with the in vivo study.

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