Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury

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1 originl rtile & 27 Interntionl Soiety of Nephrology see ommentry on pge 389 Exogenous mesenhyml stem ells lolize to the kidney y mens of CD44 following ute tuulr injury MB Herrer 1,2, B Bussolti 1,2, S Bruno 1,2, L Morndo 1, G Muriello-Romnzzi 1,2, F Snvio 3, I Stmenkovi 4, L Binone 1,2 nd G Cmussi 1,2 1 Deprtment of Internl Mediine, University of Torino, Torino, Itly; 2 Reserh Centre for Experimentl Mediine, Centro Rier Mediin Sperimentle (CeRMS), University of Torino, Torino, Itly; 3 Division of Hemtology, University of Torino, Torino, Itly nd 4 Division of Experimentl Pthology, Institut Universitire de Pthologie Universite de Lusnne, Lusnne, Switzerlnd Mesenhyml stem ells (MSC) were reently shown to migrte to injured tissues when trnsplnted systemilly. The mehnisms underlying the migrtion nd homing of these ells is, however, unler. In this study, we exmine the role of CD44 nd its mjor lignd, hyluroni id, in the trffiking of intrvenously injeted MSC in the glyerol-indued mouse model of ute renl filure (). In vitro, hyluroni id promoted dose-dependent migrtion of the stem ells tht ws inhiited y n nti-cd44 loking monolonl ntiody. In vivo, stemells injeted into mie with migrted to the injured kidney where hyluroni id expression ws inresed. Their presene orrelted with morphologil nd funtionl reovery. Renl loliztion of the MSC ws loked y pre-inution with the CD44 loking ntiody or y solule hyluroni id. Stem ells derived from CD44 knokout mie did not lolize to the injured kidney nd did not elerte morphologil or funtionl reovery. Reonstitution y trnsfetion of CD44 knokout stem ells with DNA enoding wild-type CD44, ut not loss of funtion CD44 unle to ind hyluroni id, restored in vitro migrtion nd in vivo loliztion of the ells to injured kidneys. We suggest tht CD44 nd hyluroni id intertions reruit exogenous MSC to injured renl tissue nd enhne renl regenertion. Kidney Interntionl (27) 72, ; doi:1.138/sj.ki.52334; pulished online 16 My 27 KEYWORDS: stem ells; ute renl filure; hyluronn; tuulr epithelil ells Correspondene: G Cmussi, Cttedr di Nefrologi, Diprtimento di Mediin Intern, Ospedle Mggiore S. Giovnni Bttist, Corso Dogliotti 14, 1126, Torino, Itly. E-mil: giovnni.mussi@unito.it Reeived 11 Septemer 26; revised 2 April 27; epted 1 April 27; pulished online 16 My 27 Mesenhyml stem ells (MSC) re multipotent ells present in one mrrow tht n differentite in vitro into dipoyti, hondroyti, nd osteoyti lineges. 1 In vivo MSC hve een oserved not only to regenerte tissues of mesenhyml lineges 2 4 ut lso to differentite into neurons 5 nd epithelil ells. 6 9 Aute renl filure () ssoited with nephrotoxi nd ishemi injury is most often the onsequene of ute tuulr nerosis. 1 The reovery of renl funtion following depends on pproprite replement of neroti tuulr ells with funtionl tuulr epithelium. Key plyers in kidney regenertion inlude not only mture proliferting renl ells ut lso, ording to reent reports, stem ells from lol pools s well s from the irultion Reent studies in mouse models of hve demonstrted tht MSC disply the ility to lolize in dmged kidney promoting oth morphologil nd funtionl reovery. 8,9 However, to dte, mehnisms tht underlie MSC homing to injured kidney hve not een eluidted. Tissue injury nd inflmmtion re ompnied y inresed stroml prodution of the glyosminoglyn hyluronn (HA), whih in ddition to other funtions, helps to rete low resistne highly hydrted extrellulr mtrix tht my filitte lol ellulr trffiking. 14 HA is lso undntly produed in the one mrrow y oth strom nd hemtopoieti ells 15,16 nd it is implited in the regultion of ell ell nd ell mtrix dhesion s well s in ell prolifertion nd survivl. 17,18 The proteoglyn CD44 is the prinipl ell surfe reeptor for HA. 19,2 CD44 is multifuntionl reeptor, whose stndrd isoform is expressed in hemtopoieti stem ells 21 nd MSC. 22 CD44 hs numerous funtions, inluding the regultion of ell prolifertion, differentition, survivl, migrtion into tissues, 2,21 nd hemtopoieti progenitor trffiking to the one mrrow nd spleen Bsed on these oservtions, we ddressed the role of CD44 reeptor nd its lignd HA in the reruitment of injeted MSC in mouse model of glyerol-indued. 43 Kidney Interntionl (27) 72,

2 MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells o r i g i n l r t i l e RESULTS Solule HA indued hemotti effet on CD44 þ -MSC MSC, purified from murine one mrrow, express CD44 (Figure 1). Therefore, we ddressed the possiility tht the CD44 lignd HA might hve hemotti effet on MSC nd promote their migrtion in vitro. Consistent with this notion, ddition of solule HA to the lower omprtment of trnswell stimulted migrtion of MSC through polyronte filters towrd HA (Figure 1 d). To disriminte etween the hemotti nd the hemokineti effet of HA on MSC migrtion, studies were performed in the presene of HA on oth sides of the Boyden hmer (Tle 1). The migrtory effet of HA ws relted to its grdient, suggesting hemotxis rther thn hemokinesis. Preinution of MSC with loking nti-cd44 monolonl ntiody (ma) (lone KM114) signifintly inhiited HA-indued MSC migrtion (Figure 1 nd e). Expression of HA in the renl ortex fter glyerol-indued Previous work hs shown tht in norml renl tissue, HA is detetle predominntly in the medull nd hrdly if t ll in the ortex. 26 We therefore ssessed HA expression in injured renl tissue following glyerol-indued. Intrmusulr (i.m.) injetion of glyerol indues myolysis nd hemolysis using toxi nd ishemi tuulr injury. 8 Three dys fter glyerol injetion, we oserved mrked tuulr epithelil injury, wheres ontrol mie injeted with sline lone displyed no ovious histologil hnge. 8 The lesions oserved in mie with inluded tuulr hyline st formtion, vuoliztion, nd widespred nerosis of proximl nd distl tuulr epithelium. Proximl tuules showed ytoplsmi vuoliztion, swelling nd disorgniztion of mitohondri, nd loss of the rush order. The injured ortil renl tissue ontined undnt HA deposits long the tuulr sement memrne, wheres no HA ws detetle in norml ontrols (Figure 2 nd ). Chrteriztion of CD44 / - nd CD44 þ -MSC In this study, we investigted whether CD44/HA intertion is instrumentl in the loliztion of exogenous MSC in the injured kidneys. For this purpose, we otined nd purified Tle 1 Grdient-dependent nlysis of HA-indued migrtion Upper hmer Lower hmer Vehile 2 lg/ml HA 2 lg/ml HA Vehile mg/ml HA mg/ml HA Migrtion of MSC ws performed in Boyden hmers y dding HA in the upper nd/or lower omprtments to estlish positive, negtive, sent grdient ross the filter rrier. Count 8 CD44 Migrted MSC Cells (perentge of ontrol) Control MSC+1HA MSC+2HA MSC/αCD44 + 1HA MSC/αCD44 + 2HA d e Figure 1 HA CD44 intertion indued in vitro migrtion of MSC. () FACS nlysis of CD44 þ -MSC showing expression of CD44 in 1% of ells (1 different MSC preprtions were exmined with similr results). ( e) In vitro migrtion ssy of MSC preinuted or not with 5 mg/ml loking CD44 ma (MSC/CD44) nd stimulted with HA. MSC were seeded in the upper side of the trnswell, nd HA (1 nd 2 mg/ml indited s 1HA nd 2HA) in the lower omprtment, s desried in Mterils nd methods. Migrtion ws evluted fter 18 h inution t 371C y ounting the ells tht pssed through the 8-mm memrne pores. The filters were fixed nd stined with Diff Quik. () The perentge of inresed MSC migrtion fter inution with HA in respet to ontrol hllenged with vehile lone. Vlues re expressed s % with respet to ontrol nd re the men7s.d. of three independent experiments run in triplite. ANOVA with Newmnn Keuls multiomprison test ws performed: Po.5 MSC þ 1HA nd MSC þ 2HA vs vehile; Po.5 MSC/CD44 þ 1HA nd vs MSC þ 1HA; Po.5 MSC/CD44 þ 2HA vs MSC þ 2HA. ( e) Representtive mirogrphs of MSC migrtion. Only few MSC migrted to the lower side of the memrne when hllenged with vehile lone (); severl MSC migrted to the lower side of the memrne when hllenged with 1 mg/ml HA (d) ut not when preinuted with 5 mg/ml loking CD44 ma nd hllenged with 1 mg/ml HA (e). (Originl mgnifition 2). Kidney Interntionl (27) 72,

3 o r i g i n l r t i l e MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells Counts d Counts 1 d CD44 CD44 e f e f g Figure 2 Effet of glyerol-indued tuulr injury on HA expression y renl ortex nd hrteriztion of one mrrow-derived CD44 þ -MSC nd CD44 / -MSC. () Representtive mirogrphs of ryostt setion of renl ortex from ontrol mouse killed 3 dys fter i.m. injetion of vehile lone showing sene of immunofluoresent stining for HA expression evluted with solule CD44H-humn Ig fusion protein (.5 mg/ml) (originl mgnifition 25). () Representtive mirogrphs of ryostt setion of renl ortex of mouse killed 3 dys fter i.m. injetion of glyerol showing positive immunofluoresene stining long tuulr sement memrne nd interstitium for HA expression (originl mgnifition 25). Ten mie were exmined per group with similr results. () Representtive flow ytometri nlysis of CD44 þ -MSC showing the expression of CD44. (d) Representtive flow ytometri nlysis of CD44 / -MSC showing tht MSC otined from CD44 / mie were negtive for CD44. The drk line is the test ntiody, the dotted line the isotypi ontrol. (e f) Representtive mirogrphs of dipogeni differentition of CD44 þ - nd CD44 / -MSC deteted y the presene of ft droplets. (g h) Representtive mirogrphs of osteogeni differentition deteted y positive stining for lium deposits using Alizrin Red. (Originl mgnifition f: 25). Ten different ells preprtions were tested with similr results. MSC from the one mrrow of CD44 / nd CD44 þ mie. CD44 / -MSC (t the fourth pssge) expressed the ommon mesenhyml mrkers CD15, CD29, CD73 (not shown) similrly to CD44 þ -MSC, ut not CD44 (Figure 2 nd d), s ssessed y ytofluorimetri nlysis. In ddition, MSC from CD44 / nd CD44 þ mie expressed the stem h Figure 3 Role of CD44 in renl homing of MSC in mie with glyerol-indued. MSC were deteted y immunoperoxidse stining of CFSE or y FISH nlysis of Y hromosome 24 h fter their injetion. ( ) Representtive mirogrphs showing the sene of MSC in renl setions of mouse without injeted with CD44 þ -MSC. ( d) Representtive mirogrphs showing loliztion of MSC in renl setions of mouse with injeted with CD44 þ -MSC. (e f) Representtive mirogrphs showing the sene of MSC in renl setions of mouse with injeted with CD44 / -MSC. Originl mgnifition: immunoperoxidse (, nd e) 25; FISH nlysis (, d nd f; rrows indite the positive stining for hromosome Y) 6. ell mrker Thy1 (CD9), ut not the leukoyte mrker CD45 nd CD14 or KDR, mrker of irulting endothelil progenitor ells (dt not shown). Asene of CD34 expression indited tht no ontminting hemtopoieti stem ells were present. No funtionl differenes etween CD44 / nd CD44 þ -MSC were oserved in terms of their differentition potentil towrd dipoyti (Figure 2e nd f) nd osteogeni lineges (Figure 2g nd h). However, the growth rte of CD44 / ells ws pproximtely hlf of tht of wild-type CD44 þ -MSC (dt not shown), onsistently with the reported role of CD44 in ell prolifertion. 27 Role of CD44 HA intertion in renl reruitment of MSC In the glyerol-indued model of ute renl injury, CD44 þ or CD44 / -MSC were injeted 3 dys fter glyerol dministrtion. Twenty-four hours fter injetion, CD44 þ - MSC were deteted within the renl ortex of mie with y immunoperoxidse stining of roxy-fluoresein diette, suinimidyl ester (CFSE)-lelled MSC, y fluoresene in situ hyridiztion (FISH) detetion of Y hromosome nd y iron detetion in iron-dextrn-lelled MSC (Figures 3 5). 432 Kidney Interntionl (27) 72,

4 MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells o r i g i n l r t i l e d e f Figure 4 MSC detetion y light mirosopy in mie with glyerol-indued y iron-dextrn lelling 24 h fter their injetion. ( d) Representtive mirogrphs showing loliztion of CD44 þ -MSC in renl setions of mouse with. () The presene of iron-dextrn-lelled CD44 þ -MSC in glomerulus (g; rrow) nd in perituulr pillries (rrowheds). ( nd ) The onentrtion of iron-dextrn-lelled CD44 þ -MSC round severely dmged tuules ontining protein sts. () Is prtiulr of pnel () lerly showing the interstitil loliztion of lelled MSC. (e) Presene of n isolted iron-dextrn-lelled CD44 þ -MSC (rrow) within proximl tuule. (e nd f) Asene or presene of rre iron-dextrn-lelled CD44 / -MSC (rrow) in renl setions of mouse with. The setions were stined with Prussin lue. Originl mgnifition (, d nd e) 4; () 6; (); nd (f) 25. e Figure 5 MSC detetion y eletron mirosopy in mie with glyerol-indued y iron-dextrn lelling 24 h fter their injetion. ( e) Representtive mirogrphs showing loliztion of iron-dextrn-lelled CD44 þ -MSC in renl ultrthin setions of mouse with oserved y trnsmission eletron mirosopy. () shows the presene of MSC within the lumen of glomerulr pillry. The rrow indites the iron-dextrn inlusion. ( d) show the presene of iron-dextrn-lelled CD44 þ -MSC in the perituulr pillries ( nd ) nd in the interstitium. In (), ell dherent to the endothelil lyer nd ell infiltrted in the suendothelil spe re shown. Arrows indite the iron inlusions. The insets in ( nd ) show high mgnifition of the iron-dextrn inlusions. (e) A ell ontining iron-dextrn inlusions (rrow) infiltrting proximl tuule. (f) The sene of iron-dextrn-lelled CD44 / -MSC in renl setion of mouse with. Originl mgnifition 6; insets 25. d f The lelled ells were oserved within perituulr pillries nd the interstitil spe (Figures 3 nd d, 4 nd nd 5 d). Few ells were lso deteted in the glomeruli (Figures 4 nd 5). Very few lelled ells were oserved within tuulr epithelium (Figure 4d nd 5e). No ells were deteted in ontrol mie without injeted with CD44 þ -MSC (Figure 3 nd ). In mie with injeted with CD44 / - MSC, only rre ells ould e deteted within the renl ortex (Figure 3e nd f, 4e nd f, nd 5f). The typil iron inlusions oserved y eletron mirosopy (Figure 5 nd, insets) were sent in ontrol mie, suggesting tht they were not due to uptke of iron due to hemolysis. Miniml loliztion of MSC ws oserved in the renl medull (not shown), despite the onstitutive presene of HA, suggesting tht relese of HA frgments y inflmmtion nd of other ftors produed y the injured tissue re required for MSC reruitment. To further onfirm the role of HA inding y CD44 in the erly loliztion of MSC in the injured kidney, we performed reonstitution experiments y trnsfetion of CD44 / -MSC with DNAs enoding either funtionl wildtype CD44 or the CD44R41A mutnt, tht lks the ility to ind the HA (CD44HMut) 28 (Figure 6). As expeted, fluoresein isothioynte ()-lelled HA ound CD44 þ -MSC ut not CD44 / -MSC (Figure 6 nd ). Trnsfetion of CD44 / -MSC with full-length CD44H (CD44H MSC) restored their ility to ind HA, wheres expression of CD44Hmut (CD44HMut MSC) filed to do so (Figure 6f nd g), despite CD44 ntigen expression, s ssessed y immunofluoresene (Figure 6 e). Migrtion ssy showed tht trnsfetion with full-length CD44H, ut not with CD44Hmut, restored hemotxis to HA (Figure 6h), suggesting funtionl reonstitution of CD44. Kidney Interntionl (27) 72,

5 o r i g i n l r t i l e MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells Counts Counts e h Migrted MSC (perent of ontrol) HARTC HARTC CD44+ MSC Vehile f CD44+ MSC Counts d Counts CD44 / MSC HA g CD44H MSC HARTC HARTC CD44HMut MSC Figure 6 Reonstitution of CD44 / -MSC with funtionl or non-funtionl CD44. CD44 þ -MSC, CD44 / -MSC, nd CD44 / -MSC trnsfeted with full-length CD44 DNA (CD44H) or with mutnt form of CD44 DNA (CD44HMut) trnsfetnt were generted. ( d) Representtive flow ytometri nlysis of -lelled HA-inding on CD44 þ -MSC, CD44 / -MSC nd trsfetnts. ( nd ) The -lelled HA-inding to () CD44 þ -MSC ut not () CD44 / -MSC. ( nd d) The -lelled HA-inding to () CD44H ut not to (d) CD44Hmut. The gry re is -lelled HA nd the drk line is -lelled ovine serum lumin, used s ontrol. Three different ells preprtions were exmined with similr results. (e g) Representtive mirogrphs of immunofluoresene nlysis with nti-humn CD44 (lone A3D8) ma of CD44 / -MSC trnsient trnsfeted (e) with n empty vetor, (f) with full-length CD44 DNA or (g) with mutnt form of CD44 DNA. (Originl mgnifition: 2). (h) In vitro migrtion ssy of CD44 þ -MSC, CD44 / -MSC, nd trsfetnts. MSC were seeded in the upper side of the trnswell, nd HA (1 mg/ml) in the lower omprtment, s desried in Mterils nd methods. Migrtion ws evluted fter 18 h inution t 371C y ounting the ells tht pssed through the 8-mm memrne pores. The filters were fixed nd stined with Diff Quik. Dt show the perentge of inresed MSC migrtion fter inution with HA in respet to ontrol hllenged with vehile lone. Vlues re expressed s % with respet to ontrol nd re the men7s.d. of three independent experiments run in triplite. ANOVA with Dunnett s multiomprison test ws performed Po.5 MSC stimulted with HA vs MSC stimulted with vehile. As shown in Figure 7, the perentge of lelled MSC deteted in the renl ortex 24 h fter injetion ws signifintly different in mie injeted with CD44 / -MSC in respet to CD44 þ -MSC. Moreover, CD44H MSC ut not CD44HMut MSC signifintly lolized in the injured kidneys (Figure 7), suggesting tht the inding of CD44 to HA is ritil for MSC homing. The role of CD44 in MSC loliztion ws further onfirmed y the inhiition of MSC loliztion when pre-inuted with either n nti-cd44 loking ma or solule HA (Figure 7). Role of CD44 þ -MSC in the reovery of renl injury Eight dys fter dmge (5 dys fter MSC injetion), n extensive reovery with regenertion of tuulr epithelil ells nd of the ell rush order ws oserved in mie injeted with CD44 þ -MSC (Figure 8). In ontrst, mie injeted with CD44 / -MSC displyed persistene of diffuse neroti proximl nd distl tuule injury (Figure 8). At dy 8, only sttered CD44 þ -MSC were present within the renl interstitium, wheres the CFSE-lelled nd hromosome Y-lelled ells were minly detetle within the proximl tuules (Figure 8). The numer of CFSE-positive MSC ounted in immunoperoxidse-stined setions ws % in respet to the totl numer of ounted nulei. No CD44 / -MSC were detetle t dy 8 (Figure 8d). The morphologi reovery indued y dministrtion of CD44 þ - MSC ws ompnied y reovery of the renl funtion. Blood ure nitrogen (BUN) levels tht peked t dy 3 following indution were signifintly redued t dy 8 in the nimls tht hd reeived CD44 þ -MSC (Figure 9). In ontrst, CD44 / -MSC injetion did not signifintly ffet the BUN levels, tht persisted elevted t dy 8 s in the untreted mie (Figure 9). To evlute whether the expression of CD44 ffets the severity nd the reovery of glyerol-indued, we ompred wild-type nd CD44 / mie. As shown y BUN levels, the severity of ws signifintly lower in CD44 /. However, the inresed BUN persisted t dy 8, suggesting tht the sene of reruitment of inflmmtory ells in CD44 / mie proteted mie from glyerol-indued (Figure 9), s previously desried for ishemi injury. 29 However, in these nimls, signifint reovery from dy 3 to dy 8 ws not oserved s lso inferred y histologil nlysis (not shown). To evlute whether MSC my differentite into epithelil ells, the presene of CFSE-lelled CD44 þ -MSC expressing epithelil mrkers ws deteted y fluoresene-tivted ell sorter (FACS) nlysis in ells otined from disggregted tuulr omponent of renl tissue of mie t dy 8. As shown in Figure 1, the popultion of fluoresent CFSEpositive ells oserved in CD44 þ MSC-treted nimls mounted to % of five experiments. No utofluoresent ell popultion ws deteted in ontrol mie without or with tht hd not een injeted with MSC (Figure 1). Co-stining with CFSE nd the epithelil tuulr mrkers, lens ulinris gglutinin, meglin, nd ytokertin, showed tht t lest frtion (respetively, 434 Kidney Interntionl (27) 72,

6 MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells o r i g i n l r t i l e % CFSE+ells % CFSE+ells CD MSC CD44 / - MSC CD44H - MSC CD44HMut - MSC CD44 + -MSC CD44 / -MSC CD44H-MSC CD44HMut - MSC MSC MSC MSC/Anti-CD44 MSC/HA Control Figure 7 Loliztion of CD44 þ -MSC ut not CD44 / -MSC in kidneys of mie with 24 h fter injetion. CD44 þ -MSC, CD44 / -MSC, nd CD44 / -MSC trnsfeted with full-length CD44 DNA (CD44H) or with mutnt form of CD44 DNA (CD44HMut) trnsfetnt were generted nd injeted in mie. () ount of CFSE-lelled MSC within renl tissue of ontrol mie without or of mie with glyerol-indued tht were injeted with CD44 þ -MSC, CD44 / -MSC, or CD44H MSC or CD44HMut MSC evluted 24 h fter injetion. The numer of CFSE-positive MSC ws ounted in 1 non-sequentil setions for eh experiment t 2 mgnifition nd expressed s % in respet to the totl numer of ounted nulei. Dt re expressed s men ount7s.d. of CFSE-lelled MSC deteted y immunohistohemil stining of 12 different mie. ANOVA with Newmnn Keuls multiomprison test ws performed: Po.5 þ CD44H MSC vs CD44H MSC without nd þ CD44 þ -MSC vs CD44 þ -MSC without ; 1Po.5 þ CD44 / -MSC vs þ CD44 þ -MSC; Po.5 þ CD44HMut MSC vs þ CD44H MSC. () Redution of the renl loliztion of CD44 þ -MSC 24 h fter injetion when ells were preinuted for 3 min with 5 mg/ml loking CD44 ma (MSC/CD44) or with 2 mg/ml solule HA (MSC/HA). Dt re expressed s men ount7s.d. of CFSE-lelled MSC deteted y immunohistohemil stining in 1 fields per kidney of three different mie. ANOVA with Newmnn Keuls multiomprison test ws performed: Po.5 þ MSC vs MSC; Po.5 þ MSC/CD44 vs þ MSC; Po.5 þ MSC/HA vs þ MSC. 26.2%79.65, 21.%76.8, 64.2% of five experiments) of CFSE-positive ells reovered from the kidney expressed epithelil differentition mrkers (Figure 1). Before injetion, MSC did not express ny of these epithelil mrkers (Figure 1). When CFSE-lelled CD44 / -MSC were injeted in mie, only very few fluoresent ells were deteted (Figure 1). To evlute whether MSC fused with epithelil ells in the kidney, we studied the DNA ontent of the CFSE-positive popultion y FACS nlysis. As shown in Figure 1, the CFSE-positive ells showed ploidy of 2N, rther thn 4N, inditing tht signifint fusion did not our. DISCUSSION Migrtion towrds sites of tissue injury is the first step of stem ell-medited tissue regenertion. Homing of MSC into injured tissues relies upon their ility to migrte to nd intert with the lol miroenvironment in mnner tht seures their nhorge t sites where their effetor funtions re required. We hve shown here tht expression of ell surfe CD44 is involved in the loliztion of exogenous MSC to injured renl tissue sed on the oservtions tht loss of CD44 expression y MSC or expression of CD44 loss-of-funtion mutnt results in ineffetive MSC loliztion to injured tissue nd inhiition of renl repir. Severl studies suggest tht MSC my ontriute to the reovery of ute tuulr injury. 8,9,3 It remins however unertin whether one mrrow-derived stem ells my e soure of regenerting tuulr ells or rther ontriute to tuulr reovery y mehnism of protetion. 31 Independently from the mehnism involved in renl repir, the lol reruitment of MSC is ondition neessry for their enefiil effet. 31 Currently, little is known out the moleulr mehnisms tht underlie MSC reruitment. Reent studies suggest tht triggering of the hemokine reeptor CXCR4 y its lignd stroml derived ftor my ply n importnt role in the migrtion of trnsplnted MSC to sites of injury in the rin, 32 even though CXCR4 ppers to e expressed t low level on the surfe of MSC. 33 Although it is likely tht severl mehnisms, inluding hemokine - hemokine reeptor intertions nd possily severl dhesion reeptor-lignd pirs, prtiipte in MSC homing, CD44 HA intertion my provide dominnt fore in guiding MSC to pproprite repir/regenertion sites. The importne of CD44-medited ell intertion with HA for the regultion of ell migrtion hs een shown in severl iologil proesses, inluding inflmmtion nd tumor metstsis Reently, it hs een shown tht CD44 HA intertion is involved in MSC migrtory pity. 37 In line with this study, we found tht HA-medited in vitro migrtion of MSC is prevented y preinution with n nti-cd44 loking A or solule HA. HA, the prinipl CD44 lignd, 19 hs een suggested to drive reruitment of hemtopoieti stem nd progenitor ells to the one mrrow. 23,24 However, the potentil role of CD44 in MSC homing to injured tissue hd so fr not een ddressed. HA is rely detetle in mny norml tissues in the resting stte Kidney Interntionl (27) 72,

7 o r i g i n l r t i l e MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells Figure 8 Role of CD44 þ -MSC in morphologil renl reovery in mie 8 dys fter indution. Mirogrphs re representtive of glyerol-indued ute tuulr injury fter CD44 þ -MSC nd CD44 / -MSC injetion. ( nd ) Morphologil reovery of renl dmge nd CD44 þ -MSC renl loliztion in mie with injeted i.v. with CD44 þ -MSC 3 dys fter reeiving the i.m. injetion of glyerol nd killed 5 dys fter the MSC dministrtion (dy 8). Representtive mirogrphs of light mirosopy setion stined with hemtoxylin eosin (: originl mgnifition 25) nd of eletron mirosopy (inset: originl mgnifition 4) showing the reovery of tuulr injury. () Detetion of CFSE-lelled CD44 þ -MSC y immunohistohemistry (originl mgnifition 25) nd y detetion of Y hromosome y FISH (inset: originl mgnifition 6) showing the presene of CFSE-positive nd Y hromosome-positive ells within some tuules (rrows; T ¼ tuule). ( nd d) morphologil ltertions nd CD44 / -MSC renl loliztion in mie with injeted i.v. with CD44 / -MSC 3 dys fter reeiving the i.m. injetion of glyerol nd killed 5 dys fter the MSC dministrtion (dy 8). Representtive mirogrphs of light mirosopy setion stined with hemtoxylin eosin (: originl mgnifition 25) nd of eletron mirosopy (inset: originl mgnifition 4) showing the persistene of severe tuulr injury. (d) Asene of loliztion of CFSE-lelled CD44 / -MSC y immunohistohemistry (i: originl mgnifition 25). Twelve mie were exmined per group with similr results. nd, wheres it is undnt in the renl medull, it is virtully sent from the ortex of norml kidney. 26 However, tissue remodelling ssoited with development, inflmmtion, repir, nd ner invsion is ompnied y roust indution of stroml HA prodution nd deposition. Aordingly, 3 dys fter indution of y glyerol injetion, HA deposition ws redily detetle in the interstitium nd long the tuulr sement memrne in renl ortex. Similr oservtions hve een mde following indution of ishemi-reperfusion injury. 4 The migrtory effet of solule HA on MSC tht we oserved in this study ws relted to its grdient, suggesting hemotti mehnism. During injury, it is oneivle tht protese-indued mtrix degrdtion my relese solule HA frgments tht my ontriute to the reruitment of MSC. In this study, we demonstrte tht loliztion of exogenous MSC to injured renl tissue is dependent on the CD44 HA intertion. The CD44 þ -MSC were detetle within perituulr vessels nd the interstitium 24 h fter injetion, onsistent with other studies tht demonstrted the d erly reruitment of MSC to the kidney. 41 In ontrst, MSC derived from CD44 / mie filed to lolize to the injured tissue. Disruption of CD44/HA intertion y pre-inution with nti-cd44 ma or solule HA onfirmed the relevne of funtionl CD44 for MSC reruitment. Indeed, trnsfetion of CD44 / MSC with funtionl CD44, ut not with CD44 loss-of-funtion mutnt, restored the reruitment of MSC. This result, together with experiments of CD44 lokde, indites tht CD44 hs ritil role in the loliztion of exogenous MSC in the kidney. However, we nnot exlude tht extrrenl events my ontriute to the redued renl umultion of CD44 / ells. The reruitment of MSC within the injured kidney elerted the funtionl nd morphologil reovery. Using whole-one mrrow trnsplnttion in the mouse, Poulsom et l. 42 demonstrted tht one mrrow-derived ells ould ontriute to regenertion of the renl tuulr epithelium. It is deted whether one-mrrow-derived stem ells ontriute to renl reovery y fusion or trnsdifferentition into tuulr epithelil ells. Duffield et l. 43 showed tht one mrrow does ontin ells ple of proteting the kidney from ishemi injury, ut these ells were not diretly inorported into the repired tuules. Lin et l. 44 found tht only minority of one mrrow-derived ells were inorported into the injured tuules s epithelil ells. Broekem et l. 45 demonstrted tht the tuulr engrftment of one mrrow-derived ells depends on the severity of renl dmge fter ishemi/reperfusion injury nd tht one engrfted these ells quire n epithelil phenotype. The results of this study showed tht fter 24 h, the injeted MSC were minly detetle within the perituulr pillries nd interstitium, wheres fter 8 dys MSC were found minly within tuules. The experiments with CFSE-lelled MSC followed y their reovery from the tissue indite tht out 2 2.5% of the ells reovered from the renl ortex t dy 8 derived from the injeted MSC. Fng et l. 46 found some evidene for ell fusion etween resident renl tuulr ells nd one mrrow-derived ells, ut this ws infrequent nd the signifine nd onsequenes of ell fusion in the kidney re still unler. In this study, we found tht CFSE-positive ells showed ploidy of 2N rther thn 4N, suggesting tht signifint fusion did not our. Moreover, the numer of MSC detetle in the renl injured tissue ws not inresed t 8 dys in respet to the 24 h, suggesting minor role of MSC in the repopultion of tuules. These dt re onsistent with previously reported prominent role of prolifertion of resident ell in the repopultion of tuules Owing to their low numer, the repopultion of tuules nnot e sried to the trnsdifferentition of the injeted MSC ut rther to enefiil prrine effet. MSC my fvor dedifferentition nd prolifertion of the surviving tuulr ells or stimultion of resident stem ells. 11,31 Moreover, MSC possess n nti-inflmmtory 31 nd immunosuppressive potentil 47,48 tht my e involved in the protetion of tissue injury. On the other hnd, the ility of one-mrrowderived stem ells to trnsdifferentite nd ontriute to 436 Kidney Interntionl (27) 72,

8 MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells o r i g i n l r t i l e MSC injetion BUN (mg/dl) CD44 / -MSC +CD44+ -MSC 3 8 +CD44 / -MSC +CD44+ -MSC 3 8 wt CD44 / wt CD44 / Figure 9 Evlution of renl funtion in wild-type mie injeted with CD44 þ or CD44 / -MSC, nd in CD44 / mie 3 nd 8 dys fter glyerol-indued. () Evlution of BUN in CD44 þ -MSC, CD44 / -MSC, nd MSC untreted mie efore, 3 nd 8 dys fter glyerol injetion. MSC (rrows) were injeted t dy 3. Injetion of CD44 þ -MSC ut not of CD44 / -MSC enhned the reovery of the renl funtion. Eh group onsisted of 12 mie. Dt re expressed s men7s.d. nd ANOVA with Newmnn Keuls multiomprison test ws performed: Po.5 þ CD44 þ -MSC vs þ CD44 / -MSC. () Comprison of the BUN levels in wild-type (wt) or CD44 / mie efore, 3 nd 8 dys fter glyerol injetion. The inrese of BUN in CD44 / mie ws signifintly lower then in wild-type mie t dy 3. At dy 8, the spontneous reovery ws signifint in wild-type mie ut not in CD44 / mie. Eh group onsisted of 12 mie. Dt re expressed s men7s.d. nd ANOVA with Newmnn Keuls multiomprison test ws performed: Po.5 CD44 / mie vs wt mie; Po.5 dy 8 vs dy 3. podoyte regenertion hs een reently shown in murine model of Alport syndrome. 49,5 Tissue injury of rod rnge of etiologies is ssoited with HA overprodution nd CD44 is not only funtionl in MSC ut it is expressed in most leukoyte popultions. Therefore, CD44 my lso provide mehnism for leukoyte reruitment to sites of injury with potentilly detrimentl onsequenes. 29 This is onsistent with the finding tht CD44 / mie re proteted from ishemi renl injury 29 nd, s oserved herein, from glyerol-indued. The redued severity of possily depends on the inhiition of the reruitment of inflmmtory ells. The results of this study indite tht therpeuti dministrtion of exogenous MSC tkes dvntge of the sme moleulr mehnism involved in the umultion of the inflmmtory ells within the kidney for their loliztion t the site of tissue injury. However, it is unler whether endogenous MSC exploit CD44/HA intertion in the loliztion t the site of injury. Tken together, our oservtions hve unovered n essentil role of CD44-medited HA inding in the reruitment to injured renl tissue of injeted exogenous MSC leding to n enhned renl regenertion. MATERIALS AND METHODS Mie Adult CD44 knokout mie (CD44 / ) on C57BL/6 kground (Cd44tm1Hg/J), 51 nd dult F2 Hyrid mie (C57BL/6J, 129S1/ SvImJ) were purhsed from Jkson Lortory (Br Hror, MI, USA). Studies were onduted in ordne with the Ntionl Institute of Helth Guide for the Cre nd Use of Lortory Animls. Isoltion nd ulture of MSC CD44 þ -MSC nd CD44 / -MSC from femurs nd tiis of 8-week-old mie were otined s desried previously. 52 Cells hd typil spindle-shped pperne, nd the MSC phenotype ws onfirmed y expression of MSC mrkers nd y ility to differentite into osteoytes nd dipoytes, s desried. 1 Cell migrtion ssy Twenty-four trnswell units were used for monitoring in vitro ell migrtion using 8-mm pore polyronte filters (CoStr Corp., Cmridge, MA, USA) s desried previously. 53 CD44 þ -MSC (5 1 4 ells/well in -minimum essentil medium (-MEM)), untreted or treted previously for 3 min t 371C with the loking rt nti-mouse CD44 ma (5 mg/ml, lone KM114) (Beton Dikinson, Sn Jose, CA, USA) 54 were pled in the upper hmer of the trnswell unit. -MEM with 2 or 1 mg/ml HA (from Rooster om; Sigm, St Louis, MO, USA) ws pled in the lower hmer of the trnswell unit. CD44 þ -MSC were llowed to migrte for 18 h through the memrne pores. In seleted experiments, different omintions of HA (2 nd 2 mg/ml) were dded ove nd elow the trnswell memrne. CD44 / -MSC trnsient trnsfetion Trnsient MSC trnsfetnts were generted y eletroportion t 25 V nd 9 mf in 4 mm eletroportion uvettes in BIO-Rd Kidney Interntionl (27) 72,

9 o r i g i n l r t i l e MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells SSC-Height 1 CD44 + MSC CFSE % SSC-Height vehile Control vehile SSC SSC CD44 / MSC CFSE CFSE CFSE Cytokertin Isotypi ontrol MSC efore Injetion SSC-Height SSC-Height SSC-Height SSC-Height Lens ulinris gglutinin Meglin Cytokertin MSC fter Injetion SSC-Height Gted CD44 + MSC CFSE 36% 26% 85% Lens ulinris Meglin Cytokertin gglutinin PI PI CD44 + MSC CFSE vehile Figure 1 FACS nlysis of CFSE-lelled MSC reovered from renl tissue of mie. () Column 1: representtive flow ytometri nlysis of renl ells derived t dy 8 from mouse injeted with CFSE-lelled CD44 þ -MSC showing the presene of popultion (2.5%) of fluoresent CFSE-positive ells (). Cytokertin stining () showed tht lmost ll of the CFSE-positive ells were lso ytokertin positive. Column 2 nd Column 3: representtive flow ytometri nlysis of renl ells derived from ontrol mouse without (Column 2) or with (Column 3) injeted with vehile lone, showing no uto-fluoresent ell popultion. Cytokertin () ws more expressed in ells from helthy mie thn in mie, owing to ell dmge; no doule positive ( nd ) ells were detetle. Column 4: representtive flow ytometri nlysis of renl ells derived from mouse injeted with CFSE-lelled CD44 / -MSC showing tht only very few fluoresent ells were detetle. Cytokertin stining () showed tht in mie injeted with CFSE-lelled CD44 / -MSC only few doule-positive ells were detetle. The isotypi ontrols show the sene of uto-fluoresent ells. Five experiments were performed. (): representtive flow ytometri nlysis of the expression of epithelil mrkers y CD44 þ -MSC efore nd fter the injetion in mie. In sl ondition, MSC did not express the epithelil tuulr mrkers lens ulinris gglutinin, meglin, nd ytokertin. Eight dys fter the CFSE-positive ells (gte) showed o-stining with the epithelil tuulr mrkers lens ulinris gglutinin, meglin, nd ytokertin, inditing tht t lest frtion of CFSE-positive ells expressed epithelil differentition mrkers. Five experiments were performed. () DNA nlysis of ells derived t dy 8 from mouse injeted with CFSE-lelled CD44 þ -MSC or vehile. CFSE-positive ells present in mie treted with MSC showed 2N DNA ontent. Three experiments were performed with similr results. 438 Kidney Interntionl (27) 72,

10 MB Herrer et l.: CD44-dependent renl reruitment of mesenhyml stem ells o r i g i n l r t i l e Gene-Pulser II Eletroportion System (Bio-Rd, Herules, CA, USA) with 3 mg of the pcdm8 plsmid vetor ontining full-length CD44H DNA or CD44R41A mutnt of CD44H (CD44HMut) tht nnot ind HA. 28 Cells were then plted nd used for experiments 72 h fter trnsfetion. Trnsfetion effiieny ws similr for oth onstruts rnging etween 25 nd 3% positive ells y immunofluoresene. Murine model of nd MSC trnsplnttion To evlute the ility of CD44 þ -MSC, CD44 / -MSC nd trnsfeted CD44 / -MSC to lolize into injured kidney, we indued y i.m. injetion of glyerol (Sigm) 7.5 ml/kg 8 nd, 3 dys fter glyerol injetion, MSC were intrvenously (i.v.) injeted into the til vein. CD44 þ -MSC, pre-inuted with either n nti- CD44 loking ma (KM114, 5 mg/ml) or solule HA (2 mg/ml) efore injetion, CD44 / -MSC nd trnsfeted CD44 / -MSC (pssge III IV) were hrvested using non-enzymti ell dissoition solution (Sigm). MSC were lelled with 1 mm of CFSE tht rets with the intrellulr mmines forming fluoresent onjugtes (Vyrnt ell trer kit, Moleulr Proes, Eugene, OR, USA) y inution in -MEM for 3 min s desried previously. 41 In seleted experiments, MSC were lelled for 24 h with roxydextrn-oted iron oxide nnoprtiles (Resovist, Shering, Berlin, Germny). 3 MSC were ounted, resuspended in -MEM (1 1 6 in 15 ml -MEM) nd i.v. injeted in glyerol-treted nd untreted nimls. Mie were killed 24 h or 5 dys fter trnsplnttion. Loliztion of iron-lelled MSC ws studied only t 24 h euse in preliminry experiments, we found tht fter 3 dys iron is dismissed form MSC nd n e ptured y tuulr ells. As ontrol, femle mie were injeted i.v. with sline 3 dys fter the glyerol injetion. Injeted ells were identified using immunohistohemistry with n nti-cfse polylonl A (pa), FISH (see elow), or Prussin lue to identify iron-loded ells. 3 Immunofluoresene, -lelled HA-inding ssy nd immunohistohemistry Cytofluorimetri nlysis ws performed s desried. 8. The following As were used: nti-cd15, -CD29, -CD73, -CD34, -CD45, -CD14, -Thy1 mas (Beton Dikinson); nti-cd44 ma (lone KM114); nti-ytokertin ma (Biomed, Foster City, CA, USA), nti-kdr ma (Chemion, Temeul, CA, USA), ntimeglin pa (Snt Cruz Biotehnology, Snt Cruz, CA, USA), lens ulinris gglutinin (Vetor Lortories In., Burlingme, CA, USA); phyoerythrin ()-onjugted got ginst rt immunogloulin G (IgG) (Sigm) pa, -got ginst mouse IgG nd ginst rit IgG (DkoCytomtion, Copenhgen, Denmrk), nd rit ginst got IgG (Snt Cruz) pas. The nlysis ws performed using FACSCliur ytometer (Beton Dikinson). A minimum of 1 ells were olleted for ll nlyses. CFSElelled MSC were deteted y FACS nlysis fter dissoition of the tuulr frtion deprived of glomeruli s desried previously. 11 For inding studies, ells were inuted with -lelled HA or -lelled ovine serum lumin t 5 mg/1 6 ells, s desried. 55 For HA detetion, kidney setions were inuted with solule CD44H-humn Ig fusion protein (.5 mg/ml) prepred s desried 19 or humn IgG1 isotypi ontrol A for 2 h, wshed in phosphte-uffered sline nd inuted with the -onjugted got ntihumn IgG pa (Sigm) (1:1) t 221C for 3 min. 56 Immunofluoresene on trnsfeted ultured MSC ws performed using the ntihumn CD44 (lone A3D8) ma (Sigm). Ethidium romide (Sigm) ws used for nuler ounterstining. Immunohistohemistry for MSC lelled with CFSE (Moleulr Proes) in injured kidney ws onduted using n nti-fluoresein/oregon green pa (Moleulr Proes), s desried. 41 The numer of CFSEpositive MSC ws ounted in 1 non-sequentil setions for eh experiment t mgnifition 2 nd expressed s % in respet to the totl numer of ounted nulei. DNA ontent of engrfted ells The nlysis of DNA ontent of kidney-migrted CFSE-lelled MSC ws performed s desried. 57 Briefly, isolted ells were wshed in phosphte-uffered sline efore 15 min fixtion in 4% prformldehyde, followed y fixtion in old etone for other 15 min. Therefter, ells were wshed in phosphte-uffered sline/ ovine serum lumin nd were inuted for 2 h t 41C with propidium iodte (5 mg/ml) (Sigm) to stin the DNA in solution ontining RNAse (2 mg/ml) (Sigm) nd,1% Tween 2. Cells smples were nlyzed on FACSn (Beton Dikinson). Fluoresene in situ hyridiztion Prffin kidney setions were hyridized with Strfish Cy3-leled mouse Y hromosome pint (Str-FISH; Cmio, Cmridge, UK) s previously desried. 42 After denturtion t 61C, slides were inuted with the dentured proe t 371C overnight, nd posthyridiztion ws performed t 371C y three rinses with 5% formmide/ 2 SSC nd then.1 SSC followed y phosphteuffered sline. Nulei were ounterstined with Hoehst dye (Sigm). Imges were tken with Confol mirosope (Crl Zeiss Interntionl, Germny). Morphologil studies Trnsmission eletron mirosopy ws performed on Krnovsky sfixed, osmium tetroxide-postfixed tissues nd emedded in epoxy resin ording to stndrd proedures. 58 Ultr-thin setions were stined with urnyl ette nd led itrte nd were exmined with Jeol JEM 11 eletron mirosope. BUN Blood smples from different groups were olleted. Renl funtion ws ssessed s BUN in heprinized lood using on Bekmn Synhrotron CX9 utomted hemistry nlyzer (Bekmn Instruments In., Fullerton, CA, USA). Sttistil Anlysis Sttistil nlysis ws performed y using the nlysis of vrine (ANOVA) with Newmnn Keuls multiomprison test. A P-vlue of o.5 ws onsidered signifint. ACKNOWLEDGMENTS This work ws supported y the Assoizione Itlin per l Rier sul Cnro (AIRC), y Itlin Ministry of University nd Reserh (MIUR) FIRB projet (RBNE1HRS5-1) nd COFIN, y Itlin Ministry of Helth (Rier Finlizzt 2), nd y Progetto S Polo nd y MIUR ex6% to BB, LB, nd GC. 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