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1 Originl Artil Enorinol Mt 7;:- pissn 9-9X ISSN glitzon, Novl Proxisom Prolirtor-Ativt Rptor γ Agonist, Attnuts Rnl Firosis Cus y Uniltrl Urtrl Ostrution in Mi Kwi-Hyun B,, *, Jung Bom So, *, Yun-A Jung, Hy-Young So, Sun H Kng, Hui-Jon Jon, J Mn L, Sungwoo L, Jung-Guk Kim, In-Kyu L,, Gwon-Soo Jung, Kun-Gyu Prk, Dprtmnt o Intrnl Miin, Kyungpook Ntionl Univrsity Shool o Miin; Ling-Eg Rsrh Cntr or Drug Disovry n Dvlopmnt or Dits n Mtoli Diss, Kyungpook Ntionl Univrsity Hospitl, Kyungpook Ntionl Univrsity Shool o Miin; Dprtmnts o Intrnl Miin, Surgry, Kimyung Univrsity Shool o Miin; Dprtmnt o Biohmistry n Cll Biology, Cll n Mtrix Rsrh Institut, Kyungpook Ntionl Univrsity Shool o Miin; Nw Drug Dvlopmnt Cntr, Dgu-Gyonguk Mil Innovtion Fountion, Dgu, Kor Bkgroun: Rnl tuulointrstitil irosis is ommon tur o th inl stg o nrly ll us typs o hroni kiny iss. Although lssi proxisom prolirtor-tivt rptor γ (PPARγ) gonists hv prottiv t on iti nphropthy, muh lss is known out thir irt ts in rnl irosis. This stuy im to invstigt possil niil ts o loglitzon, novl PPARγ gonist, on rnl irosis in mi. Mthos: W xmin th ts o loglitzon on rnl tuulointrstitil irosis in uniltrl urtrl ostrution () inu rnl irosis mi. W urthr in th rol o loglitzon on trnsorming growth tor (TGF)-signling pthwys in rnl tuulointrstitil irosis through in vivo n in vitro stuy. Rsults: Through hmtoxylin/osin n sirius r stining, w osrv tht loglitzon tivly ttnuts -inu rnl trophy n irosis. Immunohistohmil nlysis in onjuntion with quntittiv rvrs trnsription polymrs hin rtion n Wstrn lot nlysis rvl tht loglitzon trtmnt inhiit -inu uprgultion o rnl Sm- phosphoryltion, α-smooth musl tin, plsminogn tivtor inhiitor, n typ ollgn. In vitro xprimnts with rt msngil lls n NRK-9F rnl irolst lls suggst tht th ts o loglitzon on -inu rnl irosis r mit y inhiition o th /Sm signling pthwy. Conlusion: Th prsnt stuy monstrts tht loglitzon hs prottiv t on -inu rnl irosis, suggsting tht its linil pplitions oul xtn to th trtmnt o non-iti origin rnl iss. Kywors: Rnl tuulointrstitil irosis; glitzon; Trnsorming growth tor t; Uniltrl urtrl ostrution Riv: Novmr, Rvis: Jnury 7, Apt: Jnury 7 Corrsponing uthors: Kun-Gyu Prk Dprtmnt o Intrnl Miin, Kyungpook Ntionl Univrsity Shool o Miin, 8 Gukhosng-ro, Jung-gu, Dgu 9, Kor Tl: 8---9, Fx: 8---7, E-mil: kprk@knu..kr Copyright 7 Korn Enorin Soity This is n Opn Ass rtil istriut unr th trms o th Crtiv Commons Attriution Non-Commril Lins ( linss/y-n/./) whih prmits unrstrit non-ommril us, istriution, n rproution in ny mium, provi th originl work is proprly it. Gwon-Soo Jung Dprtmnt o Intrnl Miin, Kyungpook Ntionl Univrsity Shool o Miin, 8 Gukhosng-ro, Jung-gu, Dgu 9, Kor Tl: 8---7, Fx: 8---7, E-mil: min9@nvr.om *Ths uthors ontriut qully to this work.

2 B KH, t l. INTRODUCTION Th inin o hroni kiny iss (CKD) hs inrs in rnt yrs, with mor popl suring rom n stg rnl ilur []. A ommon pthologi tur o n stg kiny iss is rnl tuulointrstitil irosis hrtriz y trnsorming growth tor β ()/Sm signling-mit xtrllulr mtrix (ECM) umultion [,]. /Sm signling is mstr rgultory pthwy o proiroti gns suh s α-smooth musl tin (), plsminogn tivtor inhiitor (PAI-), n typ ollgn [,]. For this rson, muh ort hs n pl into ining n tiv strtgy or inhiiting /Sm signling to trt rnl tuulointrstitil irosis [,7]. Thizoliinions (TZDs), synthti proxisom prolirtor-tivt rptor γ (PPARγ) gonists, r otn us to mng typ its mllitus (TDM) vi rgultion o gluos n lipi mtolism [8]. TZDs lso t ivrs rng o tivitis inluing ll prolirtion, poptosis, inlmmtion, n oxitiv strss rsponss [9,]. Rnt stuis hv monstrt niil ts o TZD on vrious rnl injuris. Trtmnt with PPARγ gonists hs prottiv ts ginst oth iti n non-iti origin CKD [,]. Although glymi n lipi ontrol n ontriut to thir prottiv rnl t, rnt vin suggsts tht uprgultion o PPARγ xprssion in th kiny itsl provis itionl rnl nits y ruing -inu ECM proution, mintining pooyt numrs n untion, n rgulting inlmmtory ll iniltrtion [,]. glitzon is nw PPARγ gonist with TZD moity n sustitut pyrimiins, urrntly us to trt TDM tr omplting linil trils []. Phs III linil tril t show tht loglitzon trtmnt rsult in n pproximtly.% to.7% rs in glyt hmogloin ompr with tht o th plo [,7]. It is ministr s on-ily os n minly xrt in s, ruing onrns o lr nr unlik th lssi TZD pioglitzon. As nothr ntiiti gnt, it is nssry to vlut itionl ts o loglitzon on iti miro/mrovsulr omplitions. A rnt stuy hs provi vin on th riovsulr prottiv rol o loglitzon in th prolirtion n migrtion o vsulr lls. In th lloon injury rt mol, loglitzon-trt rts show lss nointiml ormtion in th roti rtry thn plo-trt rts. glitzon trtmnt lso ru th thromtous urn in th ort o polipoprotin E knokout mi high-t n high holstrol it [8]. Howvr, thr r no humn or niml stuis on ny potntil rnl prottiv ts o loglitzon. Th ts o loglitzon on rnl tuulointrstitil irosis hv lso not n stui. In th prsnt stuy, w vlut whthr loglitzon h ntiiroti ts on rnl tuulointrstitil iss in uniltrl urtrl ostrution () mi, mol o rnl tuulointrstitil irosis. W lso xmin th ntiiroti proprtis o loglitzon in vitro. METHODS Exprimntl sign C7BL mi wr prtrt with mg/kg loglitzon (Chung Kun Dng Phrmutil Corp., Soul, Kor) y gvg ily or ys. For -inu rnl irosis, th lt urtr o mi ws ouly ligt. ws prorm s prviously sri [9]. Atr, C7BL mi wr trt with mg/kg loglitzon y gvg or 7 ys onsutivly. Svn ys tr n loglitzon trtmnt, mi wr uthniz, n thir lt kinys wr rmov, ut in thirs, ix in % prormlhy, n ithr m in prin or histologi xmintion or rozn in liqui nitrogn or th isoltion o protin or RNA. All prours wr prorm in orn with institutionl guilins or niml rsrh []. Histologi n morphologi nlysis Histologi n morphologi nlysis ws prorm s prviously sri [9]. Histohmil stining ws prorm with hmtoxylin/osin n sirius r. Immunohistohmil stining ws prorm using primry ntiois ginst p- Sm (:; Snt Cruz Biothnology, Snt Cruz, CA, USA), (:; Sigm, St. Louis, MO, USA), PAI- (:; BD Biosins, Sn Jos, CA, USA), n typ ollgn (:; Am, Cmrig, UK), ollow y horsrish proxis-onjugt nti-mous or nti-rit immunogloulin G sonry ntiois (Dko, Glostrup, Dnmrk). Quntiition o rnl irosis ws msur s prviously sri [9]. Cll ultur NRK-9F norml rt kiny irolsts n rt msngil lls (RMCs) wr purhs rom th Amrin Typ Cultur Colltion (Mnsss, VA, USA). NRK-9F lls wr ultur in % CO/9% ir t 7 C in Dulo s moii Egl s mium (DMEM; Gio-BRL, Grn Isln, NY, USA) supplmnt with % tl ovin srum (FBS; Hylon, Logn, UT, Copyright 7 Korn Enorin Soity

3 glitzon Attnuts Rnl Firosis USA) n ntiiotis. RMCs wr ultur in % CO/9% ir t 7 C in DMEM. Th mium ws supplmnt with % FBS n. mg/ml G8. Clls wr rnr quisnt y inution or hours in mium supplmnt with.% FBS. Clls wr trt with mium ontining.% FBS with or without ( ng/ml; R&D Systms, Minnpolis, MN, USA) or hours. Clls wr inut with loglitzon ( μm) or hours. Clls wr susquntly pross or th isoltion o RNA or protin s sri low. Wstrn lot nlysis Wstrn lot ws prorm s prviously sri [9]. Mmrns wr inut with nti- (:,; Cll Signling Thnology, Dnvrs, MA, USA), nti-sm (:,; Cll Signling Thnology), nti-pai- (:,; BD Biosins), nti- (:,; Sigm), n nti-typ Ι ollgn (:,; Am) polylonl ntiois t C with gntl shking ovrnight. Antiois wr tt y horsrish proxis-link sonry ntioy (Snt Cruz) using n Enhn Chmiluminsn Wstrn Blotting Dttion Systm, in orn with th mnuturr s instrutions (Millipor, Billri, MA, USA) [9]. Th mmrn ws rlott with nti-β-tuulin ntioy (Appli Biologil Mtrils In., Rihmon, BC, Cn) to vriy qul protin loing in h ln. Dnsitomtri msurmnts o th ns wr prorm using UN-SCAN-IT igitizing sotwr (Silk Sintii Corp., Orm, UT, USA). Quntittiv rl-tim rvrs trnsription polymrs hin rtion Totl RNA isoltion n quntittiv rl-tim rvrs trnsription polymrs hin rtion (RT-PCR) ws prorm s prviously sri [9]. Primrs wr sign using AB StpOn sotwr vrsion. (Appli Biosystms, Fostr City, CA, USA) n wr s on th rlvnt squns rom Gn- Bnk s ollows: mous (GnBnk ssion NM_ 79.; sns, -CAGGCTGTGCTGTCCCTCTA- ; ntisns, -CGGCAGTAGTCACGAAGGAA- ), mous PAI- (GnBnk ssion NM_887.; sns, -AATCCCACA- CAGCCCATCA- ; ntisns, -GGACCACCTGCTGAAA- CACTTT- ), mous typ ollgn (GnBnk ssion NM_ 77.; sns, -GCCTTGGAGGAAACTTTGCTT- ; ntisns, -GCACGGAAACTCCAGCTGAT- ), mous glyrlhy -phospht hyrogns (GAPDH) (GnBnk ssion NM_88.; sns, -GAAGGGTGGAGCCAAAA G- ; ntisns, -GCTGACAATCTTGAGTGAGT- ), rt (GnBnk ssion NM_.; sns, -GCAC- TACCATGTACCCAGGCAT- ; ntisns, -TGCGTTCTG- GAGGAGCAATAA- ), rtpai- (GnBnk ssion NM_.; sns, -CACCCCTTCCAGAGTCCCATA- ; ntisns, -GCTGAAACACTTTTACTCCGAAGTT- ), rt typ ollgn (GnBnk ssion NM_.; sns, -GT- GCGATGGCGTGCTATG- ; ntisns, -TCGCCCTCCC- GTTTTTG- ), n rt GAPDH (GnBnk ssion NM_ 78.; sns, -TGCCGCCTGGAGAAACC- ; ntisns, -AGCCCAAGGATGCCCTTTAGT- ). Th houskping gn GAPDH ws us s n intrnl ontrol. In vitro trnsint trnstion n rportr ssy Trnsint trnstion n rportr ssy ws prorm s prviously sri []. Sttistil nlysis All t r xprss s th mn±sem. Anlysis o vrin ws us to vlut sttistil signiin. P vlus lss thn. wr onsir signiint. All xprimnts wr prorm t lst thr tims in triplit. RESULTS glitzon mliorts -inu rnl tuulointrstitil irosis Th ts o loglitzon on rnl tuulointrstitil irosis wr vlut using th mol. As shown in Fig. A, hmtoxylin/osin n sirius r stining show tht vhiltrt kinys xhiit prominnt rnl tuulr trophy n tuulointrstitil irosis. By ontrst, loglitzon-trt kinys show mrk ttnution o -inu tuulr trophy n tuulointrstitil irosis (Fig. A). glitzon supprsss th intrstitil xprssion o proiroti moluls Givn tht /Sm is wll-known mitor in th vlopmnt o rnl tuulointrstitil irosis, w xmin th ts o loglitzon on th lvls o Sm phosphoryltion n Sm trgt gns inluing, PAI-, n typ ollgn. Th rsults show tht positivly stin rs or phosphorylt Sm,, PAI-, n typ ollgn wr vintly inrs in th mg tuuls o kinys, ut ths wr signiintly ru y loglitzon trtmnt (Fig. B). Th ts o loglitzon on iroti gn xprssion wr Copyright 7 Korn Enorin Soity 7

B KH, t l. Typ ollgn Positiv stin r Positiv stin r Positiv stin r PAI- Control Positiv stin r Sirius r Sirius r stining r H&E Tuulr trophy Control 7 7 9 8 7 A B Fig.

(A) Rprsnttiv imgs o hmtoxylin n osin (H&E) n sirius r stining o kiny tissu stions rom ontrol () mi n mi with or without loglitzon (; mg/kg) trtmnt.

4 B KH, t l. Typ ollgn Positiv stin r Positiv stin r Positiv stin r PAI- Control Positiv stin r Sirius r Sirius r stining r H&E Tuulr trophy Control A B Fig.. Ets o loglitzon on uniltrl urtrl ostrution ()-inu rnopthologil hngs. (A) Rprsnttiv imgs o hmtoxylin n osin (H&E) n sirius r stining o kiny tissu stions rom ontrol () mi n mi with or without loglitzon (; mg/kg) trtmnt. Th numr o trophi tuuls ws trmin y msuring norml n ilt tuulr smnt mmrns in iv rnom ils o H&E stin stions unr high powr mgniition ( ). Ars o positiv stining with sirius r wr quntitt y omputr-s morphomtri nlysis. All morphomtri t wr normliz ginst th orrsponing vlus in nimls. Dt in ll r grphs r xprss s ol inrs rltiv to th (n= in h group). (B) Rprsnttiv imgs o immunohistohmil stining or, α-smooth musl tin (), plsminogn tivtor inhiitor (PAI-), n typ I ollgn in kiny tissu stions rom mi or mi with or without loglitzon ( mg/kg). Ars o positiv stining with psm,, PAI-, n typ ollgn ntiois wr quntitt y omputr-s morphomtri nlysis. All t wr xprss s th mn±sem o iv rnom ils rom h kiny stion (n= in h group). P<.; P<.; P<. vs. ; n P<.; P<.; P<. vs Copyright 7 Korn Enorin Soity

5 glitzon Attnuts Rnl Firosis urthr onirm y quntittiv RT-PCR n Wstrn lot nlysis. Consistnt with th immunohistohmil nlysis, th protin unn o PAI-,, n typ ollgn ws lowr in th kinys o mi ministr loglitzon thn in vhil-trt kinys (Fig. A). Morovr, -inu Sm phosphoryltion ws mrkly supprss in th kinys o loglitzon-trt mi (Fig. A). Th mrna xprssion lvls o ths gns in th kinys o loglitzontrt mi wr lso mrkly lowr thn in vhil-trt mi (Fig. B). glitzon inhiits proiroti gn xprssion through inhiition o /Sm signling To xmin th mhnism rsponsil or th ntiiroti ts o loglitzon, w xmin whthr loglitzon inhiits -stimult Sm signling in ultur rnl lls inluing NRK-9F lls n RMCs. As xpt, trtmnt inrs mrna n protin lvls o PAI-,, n typ ollgn, n inu Sm phosphoryltion. glitzon-trt NRK-9F lls show mrkly inhiit TGFβ-stimult proiroti gn xprssion n Sm phosphor- Control t-sm PAI- Typ ollgn β-tuulin Rltiv protin lvl Rltiv protin lvl Rltiv protin lvl PAI- Rltiv protin lvl Typ ollgn A Rltiv mrna lvl Rltiv mrna lvl PAI- Rltiv mrna lvl Typ ollgn B Fig.. Ets o loglitzon on proiroti gn xprssion in kinys o uniltrl urtrl ostrution () mi. (A) Rprsnttiv Wstrn lot nlysis o, t-sm, α-smooth musl tin (), plsminogn tivtor inhiitor (PAI-), n typ ollgn protin xprssion in kinys with or without loglitzon (; mg/kg; n= in h group). Dt r xprss s th mn± SEM o thr inpnnt xprimnts. (B) Rprsnttiv rl-tim rvrs trnsription polymrs hin rtion nlysis o, PAI-, n typ ollgn mrna xprssion in kinys with or without ( mg/kg; n= in h group). Dt in r grphs r mn±sem. P<.; P<.; P<. vs. ontrol (); n P<.; P<.; P<. vs.. Copyright 7 Korn Enorin Soity 9

protin lvl Rltiv protin lvl Rltiv protin lvl Rltiv mrna lvl Typ ollgn A Typ ollgn B Typ ollgn C Rltiv mrna lvl Rltiv mrna lvl PAI- Rltiv mrna lvl Typ ollgn Fig.

6 B KH, t l. t-sm PAI- Typ ollgn β-tuulin t-sm PAI- Typ ollgn β-tuulin Rltiv mrna lvl Rltiv protin lvl Rltiv protin lvl Rltiv protin lvl Rltiv protin lvl Rltiv mrna lvl PAI- PAI- PAI- Rltiv protin lvl Rltiv protin lvl Rltiv protin lvl Rltiv protin lvl Rltiv mrna lvl Typ ollgn A Typ ollgn B Typ ollgn C Rltiv mrna lvl Rltiv mrna lvl PAI- Rltiv mrna lvl Typ ollgn Fig.. Ets o loglitzon () on trnsorming growth tor β ()-inu proiroti gn xprssion in ultur kiny ll lins. Rprsnttiv Wstrn lot nlysis (A) o, t-sm, α-smooth musl tin (), plsminogn tivtor inhiitor (PAI-), n typ ollgn lvls n rprsnttiv rl-tim rvrs trnsription polymrs hin rtion (RT-PCR) nlysis (B) o, PAI-, n typ ollgn xprssion in -stimult NRK-9F lls. Rprsnttiv Wstrn lot nlysis (C) o, t-sm,, PAI-, n typ ollgn xprssion n rprsnttiv rl-tim RT-PCR nlysis (D) o, PAI-, n typ ollgn xprssion in -stimult rt msngil lls. Clls wr o-inut with ( ng/ml) n ( μm) tr hours srum strvtion. Dt r th mn±sem o thr inpnnt msurmnts (thr sprt xprimnts). P<.; P<.; P<. vs. ontrol (); n P<.; P<.; P<. vs. lon. D Copyright 7 Korn Enorin Soity

7 glitzon Attnuts Rnl Firosis Fol tivity ALK ALK Sm, Sm, A Fol tivity Fol tivity Fol tivity B Fig.. Ets o loglitzon () on th trnsorming growth tor β ()/Sm signling pthwy in kiny ll lins. (A) Ets o on plsminogn tivtor inhiitor (PAI-) promotr tivity in NRK-9F lls. Clls wr trt with ( ng/ml) with or without o-trtmnt ( μm) or hours (lt pnl). (B) Ets o on PAI- promotr tivity in rt msngil lls. Clls wr trt with ( ng/ml) with or without o-trtmnt ( μm) or hours (lt pnl). Clls wr o-trnst with th PAI- promotr n xprssion vtors or Sm/ (prk) n ALK (pdna) with or without trtmnt ( μm) or hours (right pnl). Dt r th mn±sem o thr inpnnt msurmnts. P<.; P<. vs. ontrol; n P<. vs. lon or vs. Sm/ n ALK. yltion (Fig. A, B). Consistnt with th ts in NRK-9F lls, loglitzon lso supprss -stimult Sm phosphoryltion, PAI-,, n typ ollgn in RMCs (Fig. C, D). To trmin whthr loglitzon-inu supprssion o proiroti gn xprssion is mit t th trnsriptionl lvl, w xmin whthr loglitzon trtmnt inhiits /Sm-stimult PAI- promotr tivity. In, loglitzon trtmnt sussully inhiit n ALK/ Sm, -stimult PAI- promotr tivity oth in NRK-9F lls n RMCs (Fig. ). Ths rsults init tht loglitzon hs n ntiiroti t through th inhiition o TGFβ-stimult Sm trnsriptionl tivity on its trgt gns. DISCUSSION Th stuy prsnt hr shows tht loglitzon trtmnt ttnuts rnl irosis in mi. glitzon inhiit -inu proiroti gn xprssion inluing PAI-,, n typ ollgn. Th ntiiroti ts o loglitzon wr ssoit with inhiition o th /Sm signling pthwy. PPARγ gonists r wily us s ntiiti gnts through improv insulin snsitivity n lipi mtolism []. On th sis o thir wi rng o mtoli nits, PPARγ gonists n mliort iti nphropthy in niml n humn stuis []. Rntly, svrl lins o vin show tht PPARγ gonists n ru ut kiny injury, initing tht thir rnl prottiv proprtis my prtilly inpnnt o mtoli tors. For instn, tivtion o PPARγ rus glomruloslrosis n poptosis vi rgultion o inlmmtion in vrious niml mols o non-iti nphropthy inluing / nphrtomy [], pssiv Hymnn nphritis [], ispltin-inu rnl mg [], n ishmi/rprusion injury []. Aitionlly, rosiglitzon, pioglitzon, n troglitzon hv rol in ruing rnl tuulointrstitil irosis in th mol [-9]. Rosiglitzon inhiits rnl tuulointrstitil irosis through inhiiting intrstitil mrophg iniltrtion, ownrgulting th xprssions o n its ownstrm trgt gns, n up-rgulting th BMP-7 xprssion []. Svrl groups xmin ntiiroti ts o pioglitzon n intrtion with ngiotnsin rptor ntgonists suh s L889 n nsrtn in th mol [7,8]. Pioglitzon n nsrtn hv itiv prottiv ts on rnl irosis, ut th synrgism twn pioglitzon n L889 is not lr [7]. Troglitzon ttnuts rnl intrstitil irosis n inlmmtion in os pnnt mnnr y own rgultion o signling pthwy in th mol o. In orn with ths inings, our prsnt stuy show tht loglitzon lso hs prottiv t on rnl tuulointrstitil irosis n -inu tuulr trophy. Furthrmor, loglitzon inhiit th xprssion o wllknown trgt gns, suh s PAI-,, n typ ollgn, s wll s its mjor tor, phosphorylt Sm, in th kinys o mi. Th /Sm signling pthwy is primry pthogni tor tht rivs glomrulr n tuulointrstitil irosis in th kiny y stimulting th synthsis o ECM moluls n y rsing ECM grtion []. Although msngil lls xprss Sm,,,, n 7, umulting vin monstrts tht Sm is minly implit in pthogni rol in -mit rnl irosis [-]. Rnt stuis show Copyright 7 Korn Enorin Soity

8 B KH, t l. tht svrl PPARγ gonists inhiit -stimult ECM proution, initing tht PPARγ gonists ttnut rnl tuulointrstitil irosis y inhiiting th /Sm signling pthwy []. Pioglitzon inhiits rnl tuulointrstitil irosis n iniltrtion o intrstitil mrophgs y rgulting trnsription o PAI-in mi [8]. Rosiglitzon trtmnt inhiits inlmmtory rtions n rnl irosis y ruing th ovrxprssion o nognous nothlin-, ylooxygns-, n in oxyortiostron tt-slt hyprtnsiv rts []. Our stuy s vin tht nothr novl PPARγ gonist, loglitzon supprsss tuulointrstitil irosis tr in mi y inhiiting /Sm signling. In onlusion, this stuy monstrts tht loglitzon hs rnoprottiv t on -inu rnl irosis through inhiition o th /Sm pthwy. Our rsults suggst tht loglitzon oul ply thrputi rol in CKD, proviing rtionl or urthr linil trils to vlut th iy o loglitzon in th trtmnt o CKD inluing rnl tuulointrstitil irosis. FLICTS OF INTEREST No potntil onlit o intrst rlvnt to this rtil ws rport. ACKNOWLEDGMENTS This work ws support y Biomil Rsrh Institut grnt, Kyungpook Ntionl Univrsity Hospitl (). ORCID Kwi-Hyun B Kun-Gyu Prk REFERENCES. Mgui El Nhs A, Bllo AK. Chroni kiny iss: th glol hllng. Lnt ;:-.. Zisrg M, Nilson EG. Mhnisms o tuulointrstitil irosis. J Am So Nphrol ;:89-.. Nngku M. Chroni hypoxi n tuulointrstitil injury: inl ommon pthwy to n-stg rnl ilur. J Am So Nphrol ;7:7-.. Mng XM, Nikoli-Ptrson DJ, Ln HY. TGF-t: th mstr rgultor o irosis. Nt Rv Nphrol ;:- 8.. Ey AA, Fogo AB. Plsminogn tivtor inhiitor- in hroni kiny iss: vin n mhnisms o tion. J Am So Nphrol ;7: Jung GS, Kim MK, Jung YA, Kim HS, Prk IS, Min BH, t l. Clustrin ttnuts th vlopmnt o rnl irosis. J Am So Nphrol ;: Won JC, Prk CY, Oh SW, L ES, Youn BS, Kim MS. Plsm lustrin (ApoJ) lvls r ssoit with iposity n systmi inlmmtion. PLoS On ;9:. 8. Dros G, Mioli P. Proxisom prolirtor-tivt rptor-gmm (PPAR-gmm) gonists on glymi ontrol, lipi proil n riovsulr risk. Curr Mol Phrmol ;: Cunr R, Riot M, DiCmpli D, Arhr DC, Khn DA, Glss CK, t l. Rgultion o ytokin xprssion y ligns o proxisom prolirtor tivt rptors. J Immunol ;8: Gun Y, Zhng Y, Bryr MD. Th rol o PPARs in th trnsriptionl ontrol o llulr prosss. Drug Nws Prspt ;:7-.. Smith SA, Listr CA, Tosln CD, Bukinghm RE. Rosiglitzon prvnts th onst o hyprglymi n protinuri in th Zukr iti tty rt. Dits Os Mt ;:-7.. M LJ, Mrntoni C, Linton MF, Fzio S, Fogo AB. Proxisom prolirtor-tivt rptor-gmm gonist troglitzon protts ginst noniti glomruloslrosis in rts. Kiny Int ;9: Routh RE, Johnson JH, MCrthy KJ. Troglitzon supprsss th srtion o typ I ollgn y msngil lls in vitro. Kiny Int ;:-7.. Guo B, Koy D, Isono M, Sugimoto T, Kshiwgi A, Hn M. Proxisom prolirtor-tivt rptor-gmm ligns inhiit TGF-t -inu irontin xprssion in glomrulr msngil lls. Dits ;:-8.. Kim JW, Kim JR, Yi S, Shin KH, Shin HS, Yoon SH, t l. Tolrility n phrmokintis o loglitzon (CKD- ), proxisom prolirtor-tivt rptor-gmm gonist: singl- n multipl-os, oul-lin, rnomiz ontrol stuy in hlthy ml Korn sujts. Clin Thr ;:89-.. Kim SG, Kim DM, Woo JT, Jng HC, Chung CH, Ko KS, t l. Eiy n sty o loglitzon monothrpy in ptints with typ its mllitus ovr -wks: mul- Copyright 7 Korn Enorin Soity

9 glitzon Attnuts Rnl Firosis tintr, rnomiz, oul-lin, prlll-group, plo ontroll tril. PLoS On ;9: Jin SM, Prk CY, Cho YM, Ku BJ, Ahn CW, Ch BS, t l. glitzon n pioglitzon s -ons to mtormin or ptints with typ its: -wk, multintr, rnomiz, oul-lin, prlll-group, tiv-ontroll, phs III linil tril with 8-wk xtnsion. Dits Os Mt ;7: Lim S, L KS, L JE, Prk HS, Kim KM, Moon JH, t l. Et o nw PPAR-gmm gonist, loglitzon, on nointiml ormtion tr lloon injury in rts n th vlopmnt o throslrosis. Athroslrosis ;: Jung GS, Jon JH, Cho MS, Kim SW, L IK, Kim MK, t l. Rnoprottiv t o gmigliptin, ipptiyl pptis- inhiitor, in strptozotoin-inu typ iti mi. Dits Mt J ;:-.. Jung GS, Kim MK, Cho MS, L KM, Kim HS, Prk YJ, t l. Th orphn nulr rptor SHP ttnuts rnl irosis. J Am So Nphrol 9;:-7.. Kim H, Hluzik M, Gvrilov O, Ykr S, Ports J, Sun H, t l. Thizoliinions improv insulin snsitivity in ipos tissu n ru th hyprlipimi without ting th hyprglymi in trnsgni mol o typ its. Ditologi ;7:-.. Pistrosh F, Pssur J, Hrrig K, Shwnk U, Gross P, Bornstin SR. Et o thizoliinion trtmnt on protinuri n rnl hmoynmi in typ iti ptints with ovrt nphropthy. Horm Mt Rs ;:9-8.. Bnigni A, Zoj C, Tomsoni S, Cmpn M, Corn D, Znhi C, t l. Trnsriptionl rgultion o nphrin gn y proxisom prolirtor-tivt rptor-gmm gonist: molulr mhnism o th ntiprotinuri t o pioglitzon. J Am So Nphrol ;7:-.. Jss CR, Bortoltto CF, Wilhlm EA, Romn SS, Prigol M, Noguir CW. Th proxisom prolirtor-tivt rptor-gmm gonist pioglitzon protts ginst ispltininu rnl mg in mi. J Appl Toxiol ;:-.. Doi S, Mski T, Arkw T, Tkhshi S, Kwi T, Nkshim A, t l. Prottiv ts o proxisom prolirtor-tivt rptor gmm lign on poptosis n hptoyt growth tor inution in rnl ishmi-rprusion injury. Trnsplnttion 7;8:7-.. Lin Q, Gu Y, M J, Lin SY. Prottion o rosiglitzon ginst rnl intrstitil lsion n its mhnism. Zhonghu Yi Xu Z Zhi ;8: Hn JY, Kim YJ, Kim L, Choi SJ, Prk IS, Kim JM, t l. PPARgmm gonist n ngiotnsin II rptor ntgonist mliort rnl tuulointrstitil irosis. J Korn M Si ;:-. 8. Higshi K, O T, Kushiym T, Hyoo T, Ym M, Suzuki S, t l. Aitiv ntiiroti ts o pioglitzon n nsrtn on xprimntl rnl irosis in mi. Nphrology (Crlton) ;: Kwi T, Mski T, Doi S, Arkw T, Yokoym Y, Doi T, t l. PPAR-gmm gonist ttnuts rnl intrstitil irosis n inlmmtion through rution o TGF-t. L Invst 9;89:7-8.. Ln HY, Chung AC. TGF-t/Sm signling in kiny iss. Smin Nphrol ;:-.. Mng XM, Hung XR, Chung AC, Qin W, Sho X, Igrshi P, t l. Sm protts ginst TGF-t/Sm-mit rnl irosis. J Am So Nphrol ;: Lsk A, Arhm DJ. TGF-t signling n th iroti rspons. FASEB J ;8:8-7.. Ohtomo S, Izuhr Y, Tkizw S, Ym N, Kkut T, vn Yprsl Strihou C, t l. Thizoliinions provi ttr rnoprottion thn insulin in n os, hyprtnsiv typ II iti rt mol. Kiny Int 7;7:-9.. B EH, Kim IJ, M SK, Kim SW. Rosiglitzon prvnts th progrssion o rnl injury in DOCA-slt hyprtnsiv rts. Hyprtns Rs ;:-. Copyright 7 Korn Enorin Soity

Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score

Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score 1. 1. 1 1 3 Tim tr lsion inution () wks wks 3 1 1 1 3 Tim tr lsion inution () i Ltrl sor. Mil Aov Blow.. 1 wk On sgmnt ov g 3. Lsion Fluoromylin Fluoromylin h CD11 Hinlim pling sor CD Aov Blow Fol lsion