Guetal.DiagnosticPathology2013,8:13 http://www.diagnosticpathology.org/content/8/1/13 RESEARCH Open Access Dramatic early event in chronic allograft nephropathy: increased but not decreased expression of MMP-9 gene Dongfeng Gu1†, Yanling Shi2†, Yanan Ding3, Xinyu Liu1 and Hequn Zou1* Abstract Objective: The infiltration ofmononuclear cells and replicationand migration ofsmooth muscle cells (SMCs) from media into theintima inthe vascular wall are the cardinal pathological changes inthe early stage of chronic allograft nephropathy (CAN). But the mechanism is unclear. Therefore weinvestigated the roleof matrix metalloproteinase 9(MMP-9) and its interaction withTGF-beta1, tubulointerstitial mononuclear cells infiltration and migration ofSMCs in theearly stage of CAN. Methods: Kidneysof Fisher (F334) rats were orthotopically transplanted intobilaterally nephrectomizedLewis (LEW) recipients. Tosuppress aninitial episode of acute rejection, rats were briefly treated withcyclosporine A (1.5 mg/kg/ day) for thefirst 10 days. Animals were harvested at12 weeks after transplantation for histological, immunohistochemistry and molecular biological analysis. Results: The expression ofMMP-9 was up-regulated in interstitium and vascular wall inthe early stage ofCAN, where there were interstitial mononuclear cells infiltration and SMCs migration and proliferation. Moreover the expression of MMP-9 were positively correlatedwith the degree of interstitial mononuclear cells infiltration, the quantity ofSMCs in arteriolar wall, and also theincreased TFG-beta1 expression inthe tubulointerstitiumand arteriolar wall. Conclusions: MMP-9 mayplay an important role inthe mechanism of pathological changes during theearlier period ofCAN. Virtual Slides:The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/ vs/1582313332832700. Keywords: Chronic allograft nephropathy (CAN), Mononuclear cells, Matrix metalloproteinase 9 (MMP-9), Transforming growth factor-beta1 (TGF-beta1), Smooth muscle cells (SMCs) Introduction infiltration of mononuclear cells and the migration of Nowadays, it is well known that long-term renal allograft smooth muscle cells (SMCs) from media into the intima survival is limited mainly by the progressive process of the vascular wall are considered to be the cardinal termed chronic allograft nephropathy (CAN) or chronic pathological changes during the early stage of CAN [1-4]. rejection. Pathological features of CAN consist of diffuse However, the mechanism is unclear. Thus, it is of great interstitial lymphocytes’ inflammation and fibrosis, glom- significancetounderstandtheunderlyingmechanism. erular mesangial matrix increase, glomerular sclerosis, Inotherinflammatorydiseasestheremodelingofextra- vascular intimal proliferation and tubular atrophy. The cellularmatrixisaprerequisiteforthemigrationofmono- nuclear and other cells into the tissue. Gelatinase B (matrix metalloproteinase-9, MMP-9) is a secreted multi- *Correspondence:[email protected] †Equalcontributors domain enzyme that is important for the remodeling of 1DepartmentofNephrology,TheThirdAffiliatedHospitalofSouthern the extracellular matrix and the migration of normal and MedicalUniversity,Guangzhou510630,People’sRepublicofChina tumor cells. It cleaves denatured collagens (gelatins) and Fulllistofauthorinformationisavailableattheendofthearticle ©2013Guetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Guetal.DiagnosticPathology2013,8:13 Page2of11 http://www.diagnosticpathology.org/content/8/1/13 typeIVcollagen,whichispresentinbasementmembranes. Surgery Intheimmunesystem,thiscleavagehelpslymphocytesand Operative procedures were performed under general otherleukocytestoenterandmovewithinthetissue[5]. anesthesia induced by ketamine (100 mg/kg body weight; MMP-9 belongs to the subfamily of MMPs that play an Ketamin 2%, Huadong-Pharma, Shanghai, China) admi- important role in tissue remodeling in normal and patho- nistered intraperitoneally. The left donor kidney was logical inflammatory processes. MMP-9 is a major se- isolated, removed, cooled and positioned orthotopically cretion product of macrophages and a component of into the host whose left renal vessels had been isolated, cytoplasmicgranulesofneutrophils[6],andisalsosecreted clamped, and the native kidney removed. Donor and by lymphocytes and fibroblasts [7] and vascular SMC [8] recipient renal artery, vein, and ureter were then anasto- uponstimulationbyinflammatorycytokines,suchasTGF- mosedend-to-endwith10–0prolenesutures.Noureteral beta1, IL-1,TNF-α, MCP-1 and RANTES, etc. Our previ- stent was used. Mean ischemia time was 25 minutes ousstudyhadshowntheroleofRANTESinCAN[9]. (ranges 22–31min). The remaining right kidney of the Recently,ithasbeendemonstratedthatMMP-9playsan recipientwasremovedonday10,atwhichtimethetrans- important role in the migration of mononuclear cells in plantedkidneywascheckedforsurgicaldamage.Ratswith the tissue of some chronic inflammatory diseases [10-12]. anyovertsignsofunsuccessfuloperationwerediscarded. MMP-9 can cleave basement membranes of blood vessel and extracellular matrix. The increase of MMP-9 and Experimentaldesign cleaved extracellular matrix segments parallels the infiltra- Ten male LEW rats received left renal transplantation tionofmononuclearcells[5].MMP-9producedbymono- orthotopically from ten male F334 donors. To suppress cytes and macrophages and plays an important role in the acute rejection, rats were treated with low dose CsA inflammation [5]. In an experimentally induced animal (1.5 mg/kg/day) for the first 10 days. Ten uninephrecto- model of delayed-type hypersensitivity (DTH), injection of mized male F334 rats and ten uninephrectomized male MMP-9leadtotheinfiltrationofleukocytes[13]. LEW ratswereusedascontrol animals. Newby et al. provided direct evidence for the partici- pation of MMP-9 in proliferation and migration of Functionalmeasurements SMCs (the process of arteriosclerosis) to the intima in At week 12, rats were put into metabolic cages and an invitro experiment of rabbit’saorta [14]. Aoyagi et al. 24 hour urine samples were collected and quantitative discovered that MMP-9 played an important role in the proteinuriawasdetermined.Atthesametime,serumand migration of SMCs to the intima of the rabbit carotid urine creatinine were measured and creatinine clearance arteriesafterballoondenudation[15]. wascalculated. AccordingtothecloserelationsbetweenMMP-9andthe migration of mononuclear cells and SMC, it is very likely Harvesting thatMMP-9isalso animportantfactorintheearlierstage At week 12, recipients and controls were sacrificed and ofCAN. the transplanted kidney removed. Only kidneys without Thus, we investigated the expression and its role of apparent complications of grafting such as pyeloneph- MMP-9 in the early stage of CAN in a standardized rat ritis or hydronephrosis were evaluated. Representative model. portions of the kidneys were snap-frozen in liquid nitro- gen and stored at −80°C for PCR analysis or fixed in Methods formalin (4%) for histological and immunohistological Animals evaluation. Animal research was approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical Histology University. Naive inbred male rats weighing 200-250 g Formalin-fixed and paraffin embedded sections were (ExperimentalanimalcenterofChina,Peking)wereused stained with hematoxylin/eosin to assess the grade of throughout the experiments. Lewis (LEW) rats acted as cellular infiltration and tubular atrophy. Periodic acid- graft recipients and Fisher (F334) rats as donors. They Schiff (PAS) reaction was used to evaluate the extent were housed under standard conditions at controlled of glomerulo- and arterio-sclerosis. Tissue sections were temperature, humidity and light/dark cycles, fed a stand- coded and examined in a blinded fashion by light mi- arddiet andhadfreeaccesstotapwater. croscopy. At least 200 glomeruli were counted in each section. Renal structural damage was scored semiquan- Drugs titatively on a scale from 0 to 3+ for interstitial cellular CyclosporineA(NovartisPharmaAG,Basel,Switzerland) infiltration, tubulopathy, glomerulopathy and arterial was dissolved in cremophor and administered subcu- intimal fibroplasia using the Banff criteria [16] and the taneously. sum ofscores(0–12+)wascalculated for eachsample. Guetal.DiagnosticPathology2013,8:13 Page3of11 http://www.diagnosticpathology.org/content/8/1/13 Table124-hoururineproteinexcretion,serumcreatinieandcreatinineclearenceintransplantandcontrolanimals N 24-hoururineproteinExcretion(g/d) Serumcreatinie(μmol/L) Creatinineclearence(ml/min/100gbw) Transplantanimals10 0.025±0.028 96.2±36.4 0.180±0.097 controls(LEW)10 0.035±0.016 74.3±4.9 0.266±0.079a controls(F334)10 0.040±0.024 68.7±6.2a 0.297±0.070b 24-hoururineproteinexcretiondidnotdifferbetweenthetransplantanimalsandF334controlsorLEWcontrols.Theserumcreatininelevelssignificantlydiffered betweentransplantanimalsandF344controls,butnotbetweentransplantanimalsandLEWcontrols.Thelevelsofcreatinineclearanceintransplantanimals weresignificantlyhigherthanthoseineitherF344controlsorLEWcontrols(aP<0.05,vs.allografts.bP<0.01,vs.allografts). Immunohistology medullaandtheconjunctionregion.Morethan60tubules Goat anti-rat polyclonal antibodies MMP-9 and TGF- in each biopsy section were observed. The positive area beta1 are purchased from Santa Cruz (U.S.A.) and for wasyellowstainingandImage-ProPlussoftwarewasused secondaryandtertiarystaining from DAKO(Denmark). toquantifytheintegratedopticaldensity.Therewerefour Theantigenontheformalin-fixedandparaffinembed- classed intensity of staining (negative vs. pale yellow vs. ded sections (2 μm) was restored by microwave, and pale brown vs. russet) and four classed staining area incubated with primary antibodies as mentioned above (staining area < 25% vs. staining area 25–50% vs. staining using the LSAB techniques. The intensity of tissue stain- area50–75%vs.stainingarea>75%).productofintensity ingforMMP-9andTGF-beta1wasevaluatedinablinded and relevant staining area were used for statistical manner by calculating the relative stained area using a analysis. computerizedpathologicalimageanalyticalsystem. Images were acquired by means of Leica DMR-X mi- croscope coupled to a Leica DC500 digital camera RNAisolation (Leica, Wetzlar, Germany) and the image analysis system TotalRNAwasextractedfromtherepresentativeportions Quantimet Q550 (Leica Imaging Systems). Ten ran- of the kidneys using Trizol (Gibco/BRL, U.S.A.), which domly selected discontinuous fields (400 ×) per kidney is based on the method described by Chomczynski and wereevaluated,includingtubulointerstitialinrenalcortex, Sacchi[17]. Figure1Therenalpathologicchangeofallograftsandcontrols.Pathologicalchangesofglomeruli(A),tubulointerstitium(B),andarteriolar wall(C)inallograftsandcontrols(D,E,andF,HEstaining×400). Guetal.DiagnosticPathology2013,8:13 Page4of11 http://www.diagnosticpathology.org/content/8/1/13 Figure2Histologychangeinallograftsandcontrols.ATheBanffsumsofrenalstructuraldamageinallograftsandcontrols.Ratsinthe transplantedgroupdevelopedahigherdegreeofrenalinterstitialfibrosis,tubulopathy,glomerulopathyandintimalproliferation.TheBanffsum ofscoresinallograftswassignificantlyhigherthanthatineitherLEWcontrolsorF344controls.BThepercentagesofinterstitialfibrosisin allograftsandcontrols(100×).TherewasnosignificantdifferencebetweenthepercentagesofinterstitialfibrosisofallograftsandeitherF344 controlsorLEWcontrols.CThequantityofinterstitialmononuclearcellsinallograftsandcontrols(400×).Thequantityofinterstitialmononuclear cellsinallograftssignificantlyincreasedcomparedtothatofeitherF344controlsorLEWcontrols.DThequantityofvascularSMCsinthecortex arteriolarwallofallografsandcontrols(×400).ThequantityofvascularSMCsinallograftswassignificantlyincreasedcomparedtothatofeither F344controlsorLEWcontrols. Representative portions of the kidneys samples were random primers (Promega, U.S.A). 1 μg of total RNA stored in −80°C. 60–100 mg frozen tissue samples were was added to 0.5 μg of primer. A reaction mixture con- homogenized in 1ml Trizol. 200 μl chloroform was taining MMLV reverse transcriptase (Promega), buffer added to each mixture. The mixture was centrifuged at solution (Promega),dNTP(Genda,Canada)inaconcen- 12,000 g for 15 min at 4°C. The supernatant was treated tration of 0.2 mM/L, and 25U recombinant ribonuclease with 0.5 ml isopropanol. The mixture was centrifuged at inhibitor (Huamei, China). The reaction allowed to 12,000 g for 10 min at 4°C. The supernatant was dis- proceedat37°Cfor60minutes,andwasstoppedbyheat- carded, and 0.75 ml 75% ethanol was added, and then ing to95°Cfor 5 minutesfollowed by coolingonice. The centrifuged at 7,500g for 5 min at 4°C. The precipitate cDNAwasstoredat−30°Cforfurtherprocedure. containing total RNA was stored at −80°C until further processing. RNA concentration was measured spec- AmplificationofspecificcomplementaryDNA(cDNA) trophotometrically. Specific cDNA products corresponding to mRNA for rat β-actin,MMP-9wereamplifiedusingthepolymerasechain Reversetranscription reaction (PCR). 1 μl of cDNA was taken for PCR, which Total RNA was transcribed to complementary DNA was performed in PCR buffer (Genda), using 0.2 mM/L (cDNA) by reverse transcription (RT) with hexamer dNTP (Genda), 1 μM/L of both primers (Genda), and Guetal.DiagnosticPathology2013,8:13 Page5of11 http://www.diagnosticpathology.org/content/8/1/13 Gelelectrophoresis The amplified PCR product was identified by electro- phoresis of 10 μl sample on 1.5% agarose gel stained with 0.5 μg/ml of ethidium bromide. Sample products were visualized by UV transillumination and the gel was photographed. Specific products were identified by size in relation to a known DNA MARKER (Takara Bio, Dalian, China) run with each gel. MMP-9 cDNA was semiquantitatively analyzed by densitometric compari- son to β-actin (internal control) from the same sample after the positive image was digitized by video for com- puterized densitometry. The results are given as a ratio ofintensityofMMP-9andβ-actinmRNA. Statisticalanalysis All data were expressed as mean ± SD. Statistic analysis wasperformedwithstatisticalsoftware(SPSS13.0,Chicago, IL),usingOne-WayANOVAandbivariatecorrelationana- lysis. Generally p values under 0.05 are considered significant. Results Functionalchanges 24-hour urine protein excretion did not differ between the transplant animals and controls. 24-hour urine pro- tein excretion of each group was given as mean ± SD. Figure3MMPmRNAexpressioninallograftsandcontrols. Analyzedbydensitometriccomparisontoβ-actin(internalcontrol) There was no significant difference between the trans- fromthesamesampleafterthepositiveimagewasdigitizedbyvideo plantedanimalsandtwo controlgroups(Table1). forcomputerizeddensitometry.Theresultsweregivenasaratioofthe intensityofMMP-9mRNAandβ-actinmRNA.TheexpressionMMP-9 Serumcreatininelevelsintransplantanimalsandcontrols mRNAwassignificantlyincreasedinallografscomparedtoeitherF344 controls(P<0.05)orLEWcontrols(P<0.05). We observed a tendency towards higher serum creatin- ine levels in transplant rats. The serum creatinine levels differed between transplant animals and F334 controls, 2.0 U Taq DNA polymerase (Genda). GeneAmp2700 but not between transplant animals and LEW controls Thermal Cycler (U.S.A) was used for amplification with (Table1). thefollowingsequenceprofile:initialdenaturationat95°C for 5 minutes followed by relevant number of cycles of three temperature PCR (denaturing, 94°C for 30 seconds; Creatinineclearanceintransplantandcontrolanimals annealing,65°Cfor30seconds;andextension,72°Cfor30 We observed a tendency towards lower creatinine clear- seconds) ending with a final extension at 72°C for 7 min- ance (ml/min/100 g bw) in transplanted rats. Creatinine utesandcoolingto4°C. clearance in transplant animals was significantly higher Theprimerssequence for than those in either F334 controls or LEW controls β-actin: (Table1). 0 0 up 5-ATGGTGGGTATGGGTCAGAAGG-3 (located atexon2), Histologicalchanges down 50-GTACATGGCTGGGGTGTTGAAGG-30 Atweek12post-transplantation,transplantratsdeveloped (located at exon 4). ahigherdegreeofmesangialexpansion,glomerulosclearo- Thelength oftheamplifiedsegmentswas270bp; sis and adhesions to Bowman’s capsule (Figure 1A), MMP-9: tubulopathy and interstitial mononuclear cells infiltra- 0 0 up 5-ATCGACTCCAGTAGACAATCC-3 (located at tion(Figure 1B), and intimal proliferation in allografts exon9) (Figure 1C). No abnormality was observed in glome- down 50-CAGAGAACTCGTTATCCAAGCG-30 ruli (Figure 1D), tubulointerstitium (Figure 1E) and (locatedatexon12) the arteries (Figure 1F) of F334 controls and LEW Thelength oftheamplifiedsegmentswas443bp. controls. Guetal.DiagnosticPathology2013,8:13 Page6of11 http://www.diagnosticpathology.org/content/8/1/13 Figure4ImmunohistochemicallocalizationofMMP-9andTGF-beta1inthekidneyofallograftsandcontrols.Immunohistochemical analysisshowedthatMMP-9andTGF-beta1stainedintubulointerstitiumandarteriolarwallsofallograftsandcontrols(AandCareMMP-9 staininginallografts,BandDarecontrols.EandGareTGF-beta1staininginallografts,FandHarecontrols). Banffsumsinallograftsandcontrols MMP-9 mRNA significantly increased in allografs com- The sum of these parameters such as glomerulopathy, pared toeither groupofcontrols (Figure 3). tubulopathy, interstitial mononuclear cell infiltration, intersitial fibrosis, and intimal proliferation given as Immunohistochemistry Banff sum of scores, was significantly higher in allografts MMP-9andTGF-beta1immunostainingwereobservedin than that in either F334 controls or LEW controls glomeruli,tubulaointerstitium,andarterialwall(Figure4A (Figure 2A). and 4C are MMP-9 staining in allografts; Figure 4B and 4D are controls. Figure 4E and 4G areTGF-beta1 staning Interstitialfibrosisinallograftsandcontrols inallografts;Figure4Fand4Harecontrols). There was no significant difference between the percen- tages of interstitial fibrosis in allografts and either F334 MMP-9expressioninthekidneysofallograftsand controls orLEW controls (Figure 2B). controlsatweek12post-transplantation The immunostaining of MMP-9 in tubulointerstitium of Thequantityofinterstitialmononuclearcellsinallografts allografts (0.325 ± 0.092) was significantly higher than andcontrols that of F344 controls (0.174 ± 0.052, P < 0.01) and LEW The quantity of interstitial mononuclear cells in allo- controls (0.121±0.047,P<0.01)(Figure 5A). grafts was significantly increased compared to that of Theimmunostaining ofMMP-9inarterial wall ofallo- controls (Figure 2C). grafts (0.095 ± 0.020) was significantly intense, compared to that of F344 controls (0.070 ± 0.010, P < 0.05) and Thequantityofvascularsmoothmusclecellsinthewallof LEWcontrols(0.049±0.010,P<0.05)(Figure5B). renalcortexarterioleinallograftsandcontrols The quantity of vascular smooth muscle cells of allo- grafts significantly increased compared to that of the TGF-beta1expressionintheallograftsandcontrolsat24 F334controls andLEW controls (Figure 2D). weeks The immunostaining of TGF-beta1 in tubulointerstitium Molecularbiology ofallografts(0.583±0.100)wassignificantlyintense,com- We observed a tendency towards higher level of MMP-9 paredtothatofF334controls(0.329±0.107,P<0.01)and mRNA expression in transplant rats. The expression of LEWcontrols(0.336±0.095,P<0.01)(Figure5C). Guetal.DiagnosticPathology2013,8:13 Page7of11 http://www.diagnosticpathology.org/content/8/1/13 Figure5TheexpressionofMMP-9andTGF-beta1inallograftsandcontrols.AExpressionofMMP-9inthetubulointerstiumofallografts andcontrols(100×)..TheimmunostainingofMMP-9intubulointerstitiumofallografts(0.325±0.093)significantlyincreasedcomparedtoeither F344controls(0.174±0.052)andLEWcontrols(0.121±0.047).BExpressionofMMP-9inthearteriolarwallofallograftsandcontrols(100×).The immunostainingofMMP-9inarteriolarwallofallograftssignificantlyincreasedcomparedtoF344control.CExpressionofTGF-beta1inthe tubulointerstiumofallograftsandcontrols(100×).TheimmunostainingofTGF-beta1intubulointerstitiumofallograftssignificantlyincreased comparedtoeitherF344controlsorLEWcontrols.DExpressionofTGF-beta1inthearteriolarwallofallograftsandcontrols(100×).The immunostainingofTGF-beta1inarteriolarwallofallograftssignificantlyincreasedcomparedtoeitherF344controlsorLEWcontrols. Theimmunostaining ofTGF-beta1inarteriolar wall of non-parameter correlation analysis (r = 0.757, P < 0.05, allografts (0.086 ± 0.017) was significantly intense than Figure7A). that of F344 controls (0.064 ± 0.010, P < 0.05) and LEW controls(0.070±0.012,P<0.05)(Figure5D). TheimmunostainingofMMP-9inrenalarteriolarwalland Correlationanalysis thequantityofvascularsmoothmusclecellsinallografts TheexpressionofMMP-9mRNAandthedegreeof We found positive correlation between the immunos- mononuclearcellsinfiltrationinallografs taining of MMP-9 in arteriolar wall and the quantity of We found positive correlation between the expression of vascular SMCs in allografts by bivariate non-parameter MMP-9mRNA anddegreeofmononuclear cellsinfiltra- correlationanalysis(r=0.741,P<0.05,Figure7B). tion in allografs by bivariate non-parameter correlation analysis (r=0.65, p<0.05, Figure6). TheimmunostainingofMMP-9andTGF-beta1inthe TheimmunostainingofMMP-9intubulointerstitiumand tubulointerstitiumofallografts thequantityofinterstitialmononuclearcellsinallografts We found positive correlation between the immunos- We found positive correlation between the immunos- taining of MMP-9 and TGF-beta1 in tubulointerstitium taining of MMP-9 in tubulointerstitium and the quantity of allografts by bivariate non-parameter correlation ana- of interstitial mononuclear cells in allografts by bivariate lysis(r=0.810,P<0.01,Figure7C). Guetal.DiagnosticPathology2013,8:13 Page8of11 http://www.diagnosticpathology.org/content/8/1/13 Figure6ThecorrelationbetweentheexpressionofMMP-9mRNAanddegreeofmononuclearcellsinfiltrationinallografts.MMP-9 mRNAlevelofallograftswasdemonstratedbyMMP-9mRNA/β-actinmRNAratio,thedegreeofinterstitialmononuclearcellinfiltrationwas evaluatedaccordingtoBanffCriteria,asfollow,0=Noortrivialinterstitialinflammation;1=upto25%ofparenchymainflamed;2=26%to50% parenchymainflamed;3=>50%parenchymainflamed.Theresultofbivariatenon-parametercorrelationanalysisdemonstrated:theMMP-9 mRNAlevelsofallograftswerecorrelatedtothedegreesofinterstitialmononuclearcellinfiltration.(P<0.05,Spearmancorrelationcoefficient was0.653). TheimmunostainingofMMP-9andTGF-beta1inthe in the intima of vascular wall which are the cardinal arteriolarwallofallografts pathologicalchangesofearlystageofCAN[9,19,20]. We found positive correlation between the immunos- It was shown in our data that MMP-9 expression was taining of MMP-9 and of TGF-beta1 in the arteriolar upregulated in the early stage of CAN, at the same time wall of allografts by bivariate non-parameter correlation there was significant mononuclear cells infiltration in analysis (r=0.958,P<0.001,Figure 7D). the interstitium of allografts, and the MMP-9 expression was correlated positively to the mononuclear cell infil- tration and SMC replication. Studies in other disorders Discussion such as tumor, atherosclerosis and many inflammatory The etiology of CAN is multifactorial and involves both diseases indicate that extracellular matrix degradation immune-dependent and immune-independent factors. should be prerequisite for the migration of mononuclear Immune-dependent factors include cell-mediated im- cells and other cells in the tissue, and the ability of mune responses and antibody-mediated responses to MMP-9 to degrade components of the extracellular donor antigens. Immune-independent factors include matrix and to regulate the activity of a number of sol- donor characteristics, cold ischemia time, cytomegalo- uble proteins confers an important role in various virus infection, hypertension, dyslipidemia, and the use physiological and pathological processes [16]. Therefore, of calcineurin inhibitors (CNI), notably CsA, and more the upregulated MMP-9 in the early stage of CAN, recently Tac. In our present study, we didn’t found showed in our data, might degrade extracellular matrix, T-cell infiltration and C4d sediment, so we focus on which further aggravate the mononuclear cells infiltra- the immune-independent injury of CAN. There has tion, and lead to tubulointerstitial fibrosis and chronic Ahmedabad Tolerance Induction Protocol reported for renal failure eventually. We could conclude that MMP-9 treatment of immune injuries of CAN [18], but in death might play an important role in the early stage of CAN, of reports for non-immune injury of CAN, since the the degradation of extracellular matrix caused by in- mechanism ofwhich stillneededtobefully revealed. creased expression of MMP-9 might be essential for the In the present study, we investigated the expression of migration and proliferation of mononuclear cells in the MMP-9 and its role, with F334 to LEW transplant rat early stageofCAN. model, in the early stage (12 weeks post-transplantation) OurdatasuggestthatupregulatedMMP-9expressionin of chronic allograft nephropathy which represents the the early stage of CAN has significant correlation with most common cause of late graft failure. We also inves- SMC replication in the vascular intima of the allografts. tigated the correlation between MMP-9 expression and Bendeck MP et al. found that administration of a metallo- theinfiltrationofmononuclearcellsandSMCsreplication proteinaseinhibitorafterinjuryresultedina97%reduction Guetal.DiagnosticPathology2013,8:13 Page9of11 http://www.diagnosticpathology.org/content/8/1/13 Figure7ThepotentialroleofMMPinearlierstageofCAN.AThecorrelationbetweentheimmunostainingofMMP-9intubulointerstitium andthequantityofinterstitialmononuclearcellsinallografts.TheimmunostainingofMMP-9intubulointerstitiumwascorrelatedtothequantity ofinterstitialmononuclearcellsinallografts(P<0.05,Spearmancorrelationcoefficientwas0.757).BThecorrelationbetweenthe immunostainingofMMP-9inarteriolarwallandthequantityofvascularSMCsinallografts.TheimmunostainingofMMP-9inarteriolarwallwas correlatedtothequantityofvascularSMCinallografts(P<0.05,Spearmancorrelationcoefficientwas0.741).CThecorrelationbetweenthe immunostainingofMMP-9andTGF-beta1inthetubulointerstitiumofallografts.TheimmunostainingofMMP-9andTGF-beta1in tubulointerstitiuminallograftshavecorrelation.(P<0.01,Spearmancorrelationcoefficientwas0.810).DThecorrelationbetweenthe immunostainingofMMP-9andTGF-beta1inarteriolarwallofallografts.TheimmunostainingofMMP-9andTGF-beta1inarteriolarwallof allograftshavecorrelation.(P< 0.001,Spearmancorrelationcoefficientwas0.958). in the number of SMCs migrating into the intima in rat and, therefore, is critical for the development of arterial carotid artery [21]. Recent research also provides evidence lesions of CAN by regulating both SMC migration and thatMMP-9-deficientaorticSMCshadnotonlydecreased proliferation. the migratory activity, but also decreased the capacity to Our data showed MMP-9 expression was positively make collagen contraction compared with wild-type cells correlated to the increased TGF-beta1 expression in the [22].ChoAetal.foundadenudinginjurytothearteriesof tubulointerstitium and arteriolar wall of the allografts. It wild-type mice promoted the medial SMC replication and is well known that TGF-beta1 is a key fibrogenetic cyto- the migration of medial SMCs into the neointima, but kine involved in the pathogenesis of chronic renal allo- SMC replication was significantly lower, and SMC migra- graft dysfunction. It was demonstrated in mammary tion and arterial lesion growth were significantly impaired cancer cells derived from Tenascin-C (TN-C) - deficient inMMP-9knockoutarteries.SMCs,isolatedfromMMP-9 mice, that TGF-beta1 induce MMP-9 expression in a knockoutmousearteries,showedanimpairmentofmigra- dose-dependent manner, and this inducement was sig- tion and replication in vitro [23]. Thus, based on above nificantly enhanced by addition of TN-C. Neutralization evidence, we can reach a conclusion that upregulated with specific anti-TGF-beta1 antibody showed decreased MMP-9 expression in the early stageof CAN, shown with expression of MMP-9, indicating that TGF-beta controls our data, may breakdown vascular basement membrane the baseline MMP-9 expression by a direct autocrine Guetal.DiagnosticPathology2013,8:13 Page10of11 http://www.diagnosticpathology.org/content/8/1/13 mechanism[24].KobayashiTetal.foundMMP-9expres- Received:23October2012Accepted:13January2013 sion by fibroblasts was induced by the addition of Published:28January2013 TGF-beta1 or tumour necrosis factor (TNF)-alpha to the culture medium [25]. Palosaari H et al. found TGF-beta1 References 1. 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StellasD,ElHamidiehA,PatsavoudiE:Monoclonalantibody4C5prevents of pathological changes during the earlier period of activationofMMP2andMMP9bydisruptingtheirinteractionwith extracellularHSP90andinhibitsformationofmetastaticbreastcancer chronic allograft nephropathy, and its expression may be celldeposits.BMCCellBiol2010,11:51. inducedbyTGF-beta1. 13. AnthonyDC,MillerKM,FearnS,TownsendMJ,OpdenakkerG,WellsGM, ClementsJM,ChandlerS,GearingAJ,PerryVH:Matrixmetalloproteinase expressioninanexperimentally-inducedDTHmodelofmultiple Competinginterests sclerosisintheratCNS.JNeuroimmunol1998,87(1–2):62–72. Theauthorsdeclarethattheyhavenocompetinginterest. 14. NewbyAC,SouthgateKM,DaviesM:Extracellularmatrixdegrading metalloproteinasesinthepathogenesisofarteriosclerosis.BasicRes Authors’contributions Cardiol1994,89(Suppl1):59–70. 15. AoyagiM,YamamotoM,AzumaH,NagashimaG,NiimiY,TamakiM, HZdesignthestudy,DGandYSperformedresearchandwrotethefirstdraft HirakawaK,YamamotoK:Immunolocalizationofmatrix ofthemanuscript,XLandYDparticipatedinthestatisticalanalyses.Allthe metalloproteinasesinrabbitcarotidarteriesafterballoondenudation. authorsreadandapprovedthefinalmanuscript. HistochemCellBiol1998,109(2):97–102. 16. VandenSteenPE,DuboisB,NelissenI,RuddPM,DwekRA,OpdenakkerG: Acknowledgements BiochemistryandmolecularbiologyofgelatinaseBormatrix Wewouldliketoexpressourgratitudetoallthesubjectswhoparticipated metalloproteinase-9(MMP-9).CritRevBiochemMolBiol2002, inthestudy,whichwassupportedbyInstituteofNephrologyandUrology, 37(6):375–536. theThirdAffiliatedHospitalofSouthernMedicalUniversity,Guangzhou,P.R. 17. ChomczynskiP,SacchiN:Thesingle-stepmethodofRNAisolationby China,SupportedbyNationalNaturalScienceFund(ProjectNo.81270840), acidguanidiniumthiocyanate-phenol-chloroformextraction:twenty- andalsosupportedbyResearchFundfortheDoctoralProgramofHigher somethingyearson.NatProtoc2006,1(2):581–585. EducationofChina(ProjectNo.20124433110020). 18. PatelRD,VanikarAV,AzizFA,ShahPR,TrivediHL:Ahmedabadtolerance inductionprotocolandchronicrenalallograftdysfunction:pathologic Authordetails observationsandclinicalimplications.DiagnPathol2009,4:4. 1DepartmentofNephrology,TheThirdAffiliatedHospitalofSouthern 19. LutzJ,ZouH,LiuS,AntusB,HeemannU:Apoptosisandtreatmentof MedicalUniversity,Guangzhou510630,People’sRepublicofChina. chronicallograftnephropathywitheverolimus.Transplantation2003, 2DepartmentofNephrology,WenzhouMedicalCollege,Wenzhou325035, 76(3):508–515. People’sRepublicofChina.3DepartmentofCardiovascularMedicine, 20. ViklickyO,ZouH,MullerV,LachaJ,SzaboA,HeemannU:SDZ-RAD TheThirdAffiliatedHospitalofSouthernMedicalUniversity,Guangzhou preventsmanifestationofchronicrejectioninratrenalallografts. 510515,People’sRepublicofChina. Transplantation2000,69(4):497–502.