IN-1130, a novel transforming growth factor-b type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy

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1 originl rticle & 26 Interntionl Society of Nephrology see commentry on pge 12 IN-113, novel trnsforming growth fctor- type I receptor kinse (ALK) inhiitor, suppresses renl firosis in ostructive nephropthy J-A Moon 1, H-T Kim 1, I-S Cho 1, YY Sheen 2 nd D-K Kim 2 1 R&D Center, In2Gen Co., Ltd, Smsung Cncer Reserch Institute, Seoul Ntionl University College of Medicine, Chongno-gu, Seoul, Repulic of Kore nd 2 College of Phrmcy, Ewh Womns University, Seodemun-gu, Seoul, Repulic of Kore The trnsforming growth fctor- (TGF-) plys centrl role in the progression of renl firosis. TGF- trnsduces its signl through the ctivin receptor-like kinse (ALK). IN-113, novel smll molecule ALK inhiitor, inhiited the purified kinse domin of ALK-medited Smd3 phosphoryltion with n IC vlue of.3 nm. IN-113proved to e highly selective in pnel of 27 serine/threonine nd tyrosine kinses including p38 mitogen-ctivted protein kinse. We evluted the efficcy of IN-113 to lock renl firogenesis induced y unilterl ureterl ostruction () in rts. Either vehicle (sline) or IN-113 (1 nd 2 mg/kg/ dy) ws intrperitonelly dministered to rts for 7 nd 14 dys. Phosphorylted Smd2 (psmd2) nd mrkers of firosis were nlyzed in kidney tissues. In control kidneys, interstitil firosis including tuulr trophy, loss nd diltion, inflmmtory cell infiltrtion, nd firolst cell prolifertion ws prominent. These morphologicl chnges were notly reduced y IN-113 tretment. IN-113 decresed levels of TGF-1 messenger RNA (mrna), type I collgen mrna, nd psmd2, compred to control rts. As determined y mesuring the hydroxyproline content, totl kidney collgen mount ws incresed in control kidneys, ut significntly reduced y IN-113 tretment, which ws comprle to results of histochemicl stining for collgen. IN-113 lso suppressed the expression of -smooth muscle ctin (-SMA) nd fironectin in kidneys. Our results show tht IN-113 suppressed the firogenic process of, further underscoring the potentil clinicl enefits of IN-113 in the tretment of renl firosis. Kidney Interntionl (26) 7, doi:1.138/sj.ki.177; pulished online 23 August 26 KEYWORDS: IN-113; ALK; TGF-; Renl firosis; Correspondence: D-K Kim, College of Phrmcy, Ewh Womns University, 11-1 Dehyun-dong, Seodemun-gu, Seoul 12-7, Repulic of Kore. E-mil: dkkim@ewh.c.kr Received 13 Ferury 26; revised 16 My 26; ccepted 13 June 26; pulished online 23 August 26 Trnsforming growth fctor (TGF)- fmily hs pivotl role in the regultion of vriety of physiologicl processes. Three TGF- isoforms (TGF-1, TGF-2, nd TGF-3) re expressed in mmmls, nd ech is encoded y unique gene nd expressed in tissue-specific mnner. TGF- signls through trnsmemrne receptor serine/threonine complex tht comprises the type I nd type II receptor kinses. Once ctivted, TGF- inds to the constitutively ctive type II receptor, nd the type I receptor kinse ctivin receptor-like kinse (ALK) is susequently recruited into the complex nd is ctivted y TGF- type II receptor-medited phosphoryltion. Phosphoryltion of serine/threonine residues in the ALK susequently phosphoryltes the mjor downstrem signling molecules Smd2 nd 3 proteins. Phosphorylted Smd2 (psmd2) nd 3 form complex with Smd4. This complex trnsloctes into nucleus nd regultes the trnscription of specific genes involved in cell growth, differentition, development, nd immune response. 1 Deregultion of TGF- hs een implicted in the pthogenesis of vrious diseses including firosis, therosclerosis, nd cncer. 3,6 9 Numerous studies hve consistently indicted the role of TGF- s potent firogenic cytokine evoking pthologicl firosis in vrious orgns Attempts to lock the effects of TGF- hve contriuted to the development of molecules tht inhiit TGF- inding to its receptor including decorin, 1 16 solule chimeric TGF- receptor, nd neutrlizing ntiodies The extensive knowledge regrding TGF--medited ALK-dependent signling pthwy s n inititing point t the receptor level hs highlighted the therpeutic potentil of TGF- signling ntgonist. Recent studies hve shown tht severl smll molecule denosine triphosphte (ATP)-competitive ALK inhiitors inhiit or retrd progressive firosis in kidney, lung, nd liver We hve synthesized nd developed series of ALK inhiitors tht possess n imidzole-sed moleculr scffold, cting s competitive inhiitor of the ATP inding site of ALK. We previously showed tht IN-113, representtive molecule of our compounds, inhiited trnscription of reporter genes induced y TGF-, ut did not show significnt ctivity ginst p38 mitogen-ctivted protein 1234 Kidney Interntionl (26) 7,

2 J-A Moon et l.: IN-113 suppresses renl firosis o r i g i n l r t i c l e kinse. 26 In ddition, IN-113 effectively prevented heptic firosis in the ile duct ligted rt model (sumitted). Progressive renl disese is ssocited with interstitil firosis tht is chrcterized y monocyte infiltrtion, firolst prolifertion/differentition, nd extrcellulr mtrix (ECM) protein ccumultion. Interstitil firosis is common sequel to renl injury nd is the finl common pthwy in severl renl disorders leding to end-stge renl filure. Vrious inflmmtory meditors such s cytokines, nitric oxide, nd growth fctors influence the progression of renl firosis. 3,4,27,28 Among them, TGF- plys significnt role in the progression of renl firosis y inducing the production of ECM proteins nd decresing enzymes tht degrde excessive ECM These points hve provided the therpeutic potentil of ntgonizing the TGF- pthwy in renl firotic disorders. In this study, we evluted the efficcy of IN-113 for the tretment of renl firosis in rt unilterl ureterl ostruction () model nd demonstrted for the first time tht IN-113 is effective in the prevention nd tretment of renl firosis in rt model. RESULTS Selective inhiition of ALK y IN-113 IN-113 inhiited ALK phosphoryltion of Smd3 with n IC vlue of.3 nm (Figure 1). The selectivity of IN-113 for ALK versus other kinses ws evluted using pnel of vrious kinses (Tle 1). IN-113 inhiited ALK phosphoryltion of csein with n IC vlues of 36 nm nd inhiited p38 mitogen-ctivted protein kinse with n IC vlue of 4.3 mm, demonstrting tht IN-113 is pproximtely 1-fold more selective for ALK thn p38. IN-113 exhiited IC vlues greter thn mm for other kinses. ALK phosphoryltion of Smd3 (c.p.m.) 12, 1, IC =.3 nm [IN-113] (log M) Figure 1 Dose response curve of IN-113 for its inhiitory effects on the constitutively ctive TGF-1 type I receptor ALK (T24D)-medited Smd3 phosphoryltion. Vrious concentrtions of IN-113 were mixed with rection uffer including purified ALK protein (2 ng) nd g- 32 P-ATP. The mixtures were incuted onto the Smd3-coted Flsh-Pltes for 3 h. After incution, the rdioctivities of incorported g- 32 P-ATP into the phosphorylted sites of Smd3 y ALK were mesured. The in vitro IC vlue of IN-113 ws clculted from dose response curves generted from two experiments run in duplicte (n ¼ 4) using SigmPlot softwre. Inhiition of tuulointerstitil nephritis of rts treted with IN-113 Histologicl exmintion showed tht, in control kidneys, rchitecture ws drsticlly ltered with cute nd chronic tuulr dmges including tuulr trophy, loss nd diltion, infiltrtion of inflmmtory cells, nd development of interstitil firosis (Figure 2). Most prominent dmge ws oserved in collecting ducts nd loops of Henle in the medull re, which is chrcterized y expnsion of interstitil spce with the ccumultion of firolsts nd inflmmtory cells t dy 7. Tuulointerstitil dmge ws more prominent in oth cortex nd medull t dy 14. These chnges were notly reduced or sent in nimls treted with IN-113 (Figure 2). Decresed expression of TGF-1 in kidneys y IN-113 tretment A drstic increse in TGF- is key feture of kidney. Level of TGF-1 messenger RNA (mrna) in control kidneys ws incresed 4.4- nd 4.1-fold t dys 7 nd 14 fter ureterl ligtion, respectively, compred to shm-operted nimls (Figure 3; Po.1). IN-113 dose-dependently decresed levels of TGF-1 mrna. Suppression of phosphoryltion of Smd2 in kidneys y IN-113 We determined tht increse in TGF- in kidneys is ccompnied y the increse in psmd2. Western lotting nlysis showed tht level of psmd2 in control kidneys t dy 14 ws incresed 4-fold compred to shm-operted kidneys. IN-113 significntly decresed levels of psmd2 in Tle 1 IC vlues for IN-113 ginst vrious protein kinses Protein kinse IC (lm) Protein kinse IC (lm) ALK.36 JNK3 3 ABL1 26 MAPKAPK 41 ACV-R1 8. MST4 41 ARK 41 NEK2 41 Auror-A 41 NLK 29 B-Rf 12 p CK21 41 PAK1 41 COT 41 PDGFR- 7.8 CSK 32 PIM1 41 DAPK1 41 PRK1 41 FGF-R1 13 S6K 41 FLT3 41 SGK1 41 IGF1-R 41 SRC 41 IRAK4 41 ZAP7 41 All protein kinses were expressed s humn recominnt glutthione-s trnsferse-fusion proteins or His-tgged proteins in Sf9 insect cells y using culovirus expression system. Kinses were purified y ffinity chromtogrphy using either GSH-grose or Ni-NTA-grose. ProQinse GmBH (Freiurg, Germny) performed rdioisotopic protein kinse ssys for mesuring kinse ctivities for ll enzymes except for p38. In vitro kinse inhiition ssy of IN-113 ginst p38 ws performed in MDS Phrm services. IC vlues were mesured y testing 12 concentrtions of IN-113 in ech kinse ssy. The IC vlues for IN-113 ginst ALK were determined from n experiment run in triplicte. Csein ws used s sustrte. The IC vlues for IN-113 were determined from n experiment run in triplicte. Kidney Interntionl (26) 7,

3 o r i g i n l r t i c l e J-A Moon et l.: IN-113 suppresses renl firosis c e Dy 7-/IN mg/kg Dy 14-/vehicle Dy 7-/vehicle d Dy 7-/IN mg/kg f Dy 14-/IN mg/kg chronic renl diseses. Western lotting nlysis for -SMA demonstrted tht the level of -SMA in kidneys incresed pproximtely 3- nd 9-fold t dys 7 nd 14, respectively, compred to shm-operted nimls (Figure 6; Po.1). IN-113 decresed -SMA expression in dosedependent mnner. Immunohistochemicl stining for -SMA ws performed to loclize the expression of -SMA on kidney sections (Figure 7). In control kidneys t dy 7, elongted myofirolsts stined positively for -SMA were oserved in interstitil res, nd some -SMA-positive tuulr epithelil cells were present. The undnce nd loction of -SMA expression in kidneys treted with IN-113 t dose of 2 mg/kg/dy ws similr to tht of shm-operted nimls. In kidneys with for 14 dys, strong -SMA immunorectivity ws extensively present in tuulr nd interstitil cells, ut reduced y IN-113 tretment. * * * Figure 2 Representtive photomicrogrphs of hemtoxylin eosin-stined kidney sections from shm-operted rts, control rts, nd rts treted with IN-113. () In shm kidney t dy 7, no chnges were oserved. () Histopthologicl chnges mnifested y tuulr trophy, loss nd diltion, infiltrtion of inflmmtory cells, nd prolifertion of firolstic cells (rrow heds) were prominent in control kidneys t dy 7 fter. (c) At dy 7 fter, rts treted once dily with IN-113 (1 mg/kg) reduced the extent of interstitil nephritis nd firosis (rrowheds). (d) At dy 7 fter, histopthologicl chnges shown in control kidneys were significntly reduced or sent y IN-113 tretment (2 mg/kg). (e) Severe morphologicl chnges chrcterized y tuulr trophy nd loss, nd interstititil expnsion with cellulr infiltrtes nd firous components (rrowheds) were oserved in kidneys with for 14 dys. Note the remining trophic tuulr cells (sterisks) which re surrounded y expnded interstitium. (f) At dy 14 fter, rts treted once dily with IN-113 (2 mg/kg) reduced the extent of tuulr trophy nd loss, nd interstitil nephritis nd firosis (rrowheds) compred to control kidneys. Br ¼ mm. dose-dependent mnner (Figure 4). Levels of psmd2 in shm-operted kindeys nd kidneys treted with IN- 113 were too low to quntittively mesure t dy 7 (dt not shown). Next, we performed psmd2 immunohistochemistry on kidney sections. psmd2 immunorectivity ws wek or sent in shm-operted kidneys. Strong psmd2 immunorectivity ws present in the nuclei of tuulr nd interstitil cells in kidneys, wheres wek stining ws oserved in kidneys treted with IN-113 (Figure ). Suppression of the expression of -SMA nd myofirolsts in kidneys y IN-113 The ppernce of -smooth muscle ctin (-SMA)-positive myofirolsts is considered key event in the progression of Effect of IN-113 on the expression of type I collgen mrna nd on interstitil collgen deposition Levels of type I collgen mrna in control kidneys t dys 7 nd 14 were incresed 3.- nd 9.7-fold, respectively, compred to shm-operted nimls (Figure 8; Po.1). IN-113 tretment dose dependently decresed levels of type I collgen mrna oth t dys 7 nd 14. Totl kidney collgen content ws determined y mesuring the mount of hydroxyproline, quntittive iochemicl ssy. As shown in Figure 9, hydroxyproline contents in the control kidneys incresed to 11 nd 24% t dys 7 nd 14, respectively, compred to tht ( mg/mg protein) of norml rts t the strting point (dy ). IN-113 significntly reduced hydroxyproline contents in kidneys. We further exmined the expression of collgen on Msson s trichrome stined kidney sections. The collgen ccumultion were prominent in the interstitil spce of control kidneys, compred to shm-operted nimls (Figure 1). IN-113 mrkedly reduced the collgen ccumultion in control kidneys (Figure 1). Effect of IN-113 on totl kidney fironectin We lso investigted the effect of IN-113 on the expression of fironectin, mjor interstitil mtrix component, in kidneys (Figure 11). Compred to shm-operted nimls, kidneys exhiited 17- nd 3-fold increses in the expression of fironectin t dys 7 nd 14, respectively. IN-113 suppressed the levels of fironectin in kidneys in dose-dependent mnner. DISCUSSION We previously identified IN-113 s potent smll molecule inhiitor of TGF- signling pthwy in cell-sed ssys. The present study clerly shows tht IN-113 effectively inhiits the in vitro ALK ctivity without ffecting other serine/threonine nd tyrosine kinses exmined. Recently, severl ALK inhiitors hve een developed s potentil 1236 Kidney Interntionl (26) 7,

4 J-A Moon et l.: IN-113 suppresses renl firosis o r i g i n l r t i c l e (dy 7) (dy 14) IN IN TGF-β1 TGF-β1 HPRT HPRT Reltive TGF-β1 mrna (corrected with HPRT) # ## Reltive TGF-β1 mrna (corrected with HPRT) *, # ## 1 2 (dy 7) IN (dy 14) IN-113 Figure 3 Suppression of TGF-1 mrna expression in rt kidneys y IN-113. () Representtive reverse trnscriptse-pcr photogrphs (upper pnel) showing levels of TGF-1 mrna of kidneys from shm-operted rts, control rts, nd rts treted with IN-113 for 7 nd 14 dys. Numers 1 nd 2 indicte two individul nimls from ech group. HPRT served s n internl control. () SYBR Green rel-time PCR nlysis ws used to quntify levels of TGF-1 mrna in the kidneys of shm-operted rts, control rts, nd rts treted with IN-113 for 7 nd 14 dys. Tretment of IN-113 (1 nd 2 mg/kg/dy) significntly decresed the mount of TGF-1 mrna in control kidneys. Vlues re s.e.m. of 7 (dy 7) or 4 (dy 14) nimls per group. *Po. versus shm control group. Po.1 versus shm control group. # Po. versus control group. ## Po.1 versus control group. IN-113 (dy 14) 1 2 Vehicle c IN mg/kg psmd2 β-actin Dy 7 Reltive undnce (fold induction) , # 1 2 (dy 14) IN-113 Figure 4 Suppression of phosphoryltion of Smd2 in rt kidneys y IN-113. () Representtive Western lotting photogrph (upper pnel) showing levels of psmd2 protein in the kidneys of shm-operted rts, control rts, nd rts once dily treted with either vehicle or IN-113 for 14 dys. The sme lot ws stripped nd reproed with -ctin to confirm equl loding (lower pnel). Numers 1 nd 2 indicte two individul nimls from ech group. () Quntittive nlysis of the reltive undnce of psmd2 protein fter normliztion with -ctin. Vlues (fold induction reltive to shm-operted control) re s.e.m. of four nimls per group. *Po. versus shm control group. Po.1 versus shm control group. # Po.1 versus control group. therpy in firosis diseses. Among them, SB-2334 is smll molecule ALK inhiitor nd hs similr moleculr scffold with IN We compred the potency of IN-113 with SB-2334 nd found out tht IN-113 is *, # Dy 14 d e f Figure Representtive photomicrogrphs of psmd2 immunohistochemistry on kidney sections from shm-operted rts, control rts, nd rts treted with IN-113. ( nd d) In shm-operted nimls, no significnt nucler psmd2 stining ws oserved t () dy 7 nd (d) dy 14. ( nd e) Nucler locliztion of psmd2 in tuulr nd interstitil cells ws incresed in control kidneys t () dy 7 nd (e) dy 14 (rown, rrowheds) compred to shm-operted kidneys. (c nd f) IN-113 (2 mg/kg) tretment reduced nucler stining intensity (rown, rrowheds) in interstitil nd tuulr res of kidneys t (c) dy 7 nd (f) dy 14, which re ssocited with the decrese in the extent of interstitil nephritis nd firosis compred to control kidneys. Br ¼ 8 mm. more potent thn SB-2334 in oth enzyme inhiition nd in vitro cell-sed ssys (dt not shown). The ostructed kidneys t 7 dys fter ureterl ligtion in the present study showed typicl fetures of ostructive nephropthy such s the presence of myofirolsts nd inflmmtory cells in the interstitium, tuulr degenertion nd trophy, nd interstitil firosis. Tuulointerstitil dmges progressed in kidneys t dy 14. These morphologicl chnges re ccompnied y increses in the Kidney Interntionl (26) 7,

5 o r i g i n l r t i c l e J-A Moon et l.: IN-113 suppresses renl firosis IN-113 (dy 7) 1 2 IN-113 (dy 14) 1 2 α-sma α-sma β-actin β-actin Reltive undnce (fold induction) * *, ## Reltive undnce (fold induction) ## 1 2 IN IN-113 (dy 7) (dy 14) Figure 6 Suppression of -SMA expression in rt kidneys y IN-113. () Representtive Western lotting photogrphs (upper pnel) showing levels of -SMA protein in the kidneys of shm-operted rts, control rts, nd rts once dily treted with either vehicle or IN-113 for 7 nd 14 dys. The sme lot ws stripped nd reproed with -ctin to confirm equl loding (lower pnel). Numers 1 nd 2 indicte two individul nimls from ech group. () Quntittive nlysis of the reltive undnce of -SMA protein fter normliztion with -ctin. Vlues (fold induction reltive to shm-operted control) re s.e.m. of 7 (dy 7) or 4 (dy 14) nimls per group. *Po.1 versus shm control group. # Po. versus control group. ## Po.1 versus control group * *, # *, ## Dy 7 Dy 14 Cortex Medull Cortico-medull Vehicle c d e f g h i IN mg/kg Figure 7 Representtive photomicrogrphs of -SMA immunohistochemistry on kidney sections from shm-operted rts, control rts, nd rts treted with IN-113. (, d, nd g) In shm-operted nimls, -SMA expression (rown color) is limited to lood vessels (rrows). ( nd e) Strong immunorectivity ws oserved in interstitil nd tuulr res of control kidney t dy 7 fter. (h) The extent of -SMA intensity nd the numer of cells expressing -SMA in control kidney t dy 14 ws incresed compred to those of control kidney t dy 7 ( nd e). (c nd f) Once-dily tretment with IN-113 (2 mg/kg) for 7 nd (i) 14 dys reduced stining intensity in interstitil nd tuulr res of kidneys, which is ssocited with the decrese in the extent of interstitil nephritis nd firosis. Br ¼ 3 mm. mounts of -SMA, hydroxyproline, collgen, nd fironectin tht re specific iochemicl mrkers for firosis. Our study shows tht IN-113 lmost completely suppressed the expression of -SMA, collgen, nd fironectin in kidneys t n erly stge (dy 7), which is ccompnied y the ttenution of tuulointerstitil firosis. Although IN-113 did not confer complete protection from renl firosis in kidneys t lter stge (dy 14), IN-113 significntly inhiited firogensis in kidneys. TGF- is elieved to e the prime firogenic molecule in model. A drstic increse in TGF-1 ws oserved in kidneys. Interestingly, IN-113 tretment decresed the mount of TGF-1 mrna in kidneys eing ccompnied y the ttenution of ll firogenic fetures exmined. Decresed TGF-1 mrna expression y IN-113 my e the indirect consequence of reduced firogenic cellulr components including firolsts nd myofirolsts tht produce TGF-. Also, this reduction of TGF-1 mrna y IN-113 my contriute to less numers of inflmmtory cells such s mcrophges nd firolsts in the interstitium s TGF- is potent chemottrctnt for those cell types. 32 Indeed, we found tht IN-113 decresed the numer of infiltrting mcrophges in kidneys which were identified y immuohistochemistry using monoclonl ED1 ntiody (dt not shown). In the present study, we did not identify the cellulr source of TGF- in kidneys, ut severl reports hve indicted tht the mjor cell component tht produces TGF- in model is interstitil cells. However, contriution of tuulr cells to the incresed TGF- levels cnnot e excluded s tuulr cells re known to upregulte TGF-. 33,34 In the present study, levels of TGF-1 mrna in control kidneys were similr etween t dys 7 nd 14 fter unilterl ligtion, which is consistent with the previous reports in which, unlike other firogenic mrkers, level of TGF- ws not prominently elevted from erly to lte stges. 3, Kidney Interntionl (26) 7,

6 J-A Moon et l.: IN-113 suppresses renl firosis o r i g i n l r t i c l e (dy 7) (dy 14) IN IN Col I Col I HPRT HPRT Reltive type 1 collgen mrna (corrected with HPRT) * # # 1 2 (dy 7) IN-113 Reltive type 1 collgen mrna (corrected with HPRT) 12 * * *, # *, # 1 2 (dy 14) IN-113 Figure 8 Suppression of type 1 collgen mrna expression in control kidneys y IN-113. () Representtive reverse trnscriptse-pcr photogrph (upper pnel) showing levels of collgen type I mrna of kidneys from shm-operted rts, control rts, nd rts treted with IN-113 for 7 nd 14 dys. Numers 1 nd 2 indicte two individul nimls from ech group. HPRT served s n internl control. () SYBR Green rel-time PCR nlysis ws used to quntify levels of the collgen type 1 mrna in the kidneys of shm-operted rts, control rts, nd rts treted with IN-113 for 7 nd 14 dys. Tretment of IN-113 (1 nd 2 mg/kg/dy) significntly decresed the mount of type I collgen mrna in control kidneys. Vlues re s.e.m. of 7 (dy 7) or 4 (dy 14) nimls per group. *Po.1 versus shm control group. # Po.1 versus control group. Hydroxyproline (μg/mg protein) Hydroxyproline (μg/mg protein) *, # ## 1 2 (dy 7), #, ## 1 2 (dy 14) IN-113 IN-113 Figure 9 Effect of IN-113 on the hydroxyproline content in control kidneys. IN-113 tretment significntly decresed levels of hydroxyproline in control kidneys t () dy 7 nd() dy 14. Vlues re s.e.m. of 7 (dy 7) or 4 (dy 14) nimls per group. *Po. versus shm control group. Po.1 versus shm control group. # Po. versus control group. ## Po.1 versus control group. Dy 7 Dy 14 High power High power Low power Vehicle c d e f g h i IN mg/kg Figure 1 Representtive photomicrogrphs of Msson s trichrome stining on kidney sections from shm-operted rts, control rts, nd rts treted with IN-113. (, d, ndg) In shm-operted nimls, collgen expression is limited to lood vessels (lue, rrows). (lue in nd e) Collgen expression is prominent in interstitil nd tuulr res of control kidneys t dy 7 fter ( nd e). (lue in h) Collgen expression is present in widespred pttern t dy 14 fter. Once-dily tretment with IN-113 (2 mg/kg) for 7 (c nd f) nd 14 dys (i) reduced stining intensity in interstitil nd tuulr res of kidneys, which is ssocited with the decrese in the extent of interstitil nephritis nd firosis. Collgen is present only round lood vessels (rrows in c) in kidneys once dily treted with IN-113 (2 mg/kg) for 7 dys. Br ¼ 3 mm. This discrepncy my e explined y the oservtion tht, in the lte stge (dy 14), more thn 6 7% of renl tuulr prenchym which my produce TGF- ws replced y firotic components. IN-113 suppressed phosphoryltion of Smd2, mrker of TGF-/ALK signling ctivtion. This reduction of psmd2 is well correlted with the reductions seen for other firogenic mrkers, indicting tht psmd2 is mjor Kidney Interntionl (26) 7,

7 o r i g i n l r t i c l e J-A Moon et l.: IN-113 suppresses renl firosis IN-113 Fironectin β-actin (dy 7) (dy 14) 1 2 IN Fironectin β-actin Reltive undnce (fold induction) *, # 1 2 (dy 7) ## IN-113 Reltive undnce (fold induction) , #, ## 1 2 (dy 14) IN-113 Figure 11 Suppression of fironectin expression in control kidneys y IN-113. () Representtive Western lotting photogrph (upper pnel) showing levels of fironectin protein in kidneys of shm-operted rts, control rts, nd rts once dily treted with IN-113 for 7 nd 14 dys. The sme lot ws stripped nd reproed with -ctin to confirm equl loding (lower pnel). Numers 1 nd 2 indicte two individul nimls from ech group. () Quntittive nlysis of the reltive undnce of fironectin protein fter normliztion with -ctin. Vlues (fold induction reltive to shm control) re s.e.m. of 7 (dy 7) or 4 (dy 14) nimls per group. *Po. versus shm control group. Po.1 versus shm control group. # Po. versus control group. ## Po.1 versus control group. downstrem signling molecule in the firogenesis of kidneys. Tken together, our study demonstrtes tht the inhiition of ALK signling pthwy y IN-113 could interfere with the utocrine/prcrine effect of TGF- in firogenic nd inflmmtory cells, leding to the locking of the vicious firosis cycle in model. It should e mentioned here tht this reduction of the numer of firogenic cells is not relted to the direct effect of IN-113 on the cell cycle-relted events ecuse IN-113 did not show n ntiprolifertive effect on severl cell lines in vitro t the concentrtion of up to 1 mm (dt not shown). Myofirolsts re not present in norml kidney. The ppernce of myofirolst cells expressing -SMA protein is one of the erly chnges leding to firosis in the kidney. -SMA is considered specific for epithelil myofirolst trnsdifferentition. Elongted myofirolsts expressing -SMA were found in moderte numers in firotic interstitium of kidneys, nd some tuulr epithelil cells were stined with -SMA. These findings indicte tht there is continuous trnsition etween -SMA-positive epithelil cells nd positively stining cells with myofirolst morphology. This event is proly induced y TGF- tht is incresed during the pthogenesis of interstitil nephritis nd firosis. Myofirolsts hve een known to produce interstitil ECM components such s type I collgen nd fironectin. 37 As shown in Figures 7 nd 1, in control kidneys, type I collgen ws coloclized on the -SMA-positive cells in the interstitium. IN-113 tretment significntly reduced the expression of -SMA-positive cells, type I collgen, nd fironectin. This finding suggests tht locking of TGF- signling pthwy is sufficient to prevent the genertion of ECM components. A similr result ws previously otined when IN-113 ws used for the tretment of heptic firosis induced y ile duct ligtion in rts in which IN-113 successfully decresed levels of -SMA protein nd type I collgen (sumitted). Our study clerly demonstrtes tht the inhiition of firogenesis in kidneys y interfering TGF- signling is sufficient to prevent the firogenesis, which is consistent with the previous reports in which nother TGF- signling ntgonists including nti-tgf- ntiody could prevent the firogenic process in kidneys Tken together, the TGF- signling pthwy plys mjor role in firogenesis in kidneys even though the renl firosis is considered multi-component disese. However, it should e mentioned here tht IN-113 efficiently meliorted renl firosis, ut did not completely prevent the firogenic process in kidneys t the lte stge (dy 14). These results suggest tht renl firosis in ostructed kidneys t lte stges involve TGF--dependent s well s TGF--independent pthwys. Therefore, IN-113 could e more effective for the tretment of renl firosis when used in comintion with other nti-firotic gents tht possess different mode of ctions. TGF- singling ntgonists in the lte-stge (phse II/III) clinicl development for the tretment of firotic disorders re protein-sed molecules such s nti-tgf- ntiodies. Initil efforts hve een focused on the development of ntiody-sed TGF- ntgonists ecuse the primry trget for the TGF- signling pthwy is heterodimeric TGF- receptor kinse nd it ws thought tht it would e very difficult to develop smll molecule drug tht very specificlly inhiits the TGF- receptor kinse such s ALK. Mny smll molecule ALK inhiitors tht re eing developed possess the ATP-competitive nture nd the 124 Kidney Interntionl (26) 7,

8 J-A Moon et l.: IN-113 suppresses renl firosis o r i g i n l r t i c l e unique mechnism of ction. There re severl dvntges inherent in these smll molecule ALK inhiitors in terms of the drug delivery efficiency. Orlly ioville smll molecule ALK inhiitors my overcome the limit of tissue penetrtion nd delivery issues of nti-tgf- ntiody therpies. In conclusion, we demonstrte for the first time tht IN-113 possesses evident protective effects on renl interstitil firosis in rts. Our study provides n encourging exmple of the therpeutic potentil of modulting TGF- signling in progressive renl firosis nd is indictive of the potentil success of IN-113 in the tretment of renl firosis. MATERIALS AND METHODS DNA expression constructs nd in vitro ALK inhiition ssy Wild-type full-length ALK nd Smd3 complementry DNAs were cloned y polymerse chin rection (PCR), s descried previously. 38 The cytoplsmic region (mino cids ) of ALK ws then mplified. Constitutively ctive ALK (T24D) ws mde y mutting threonine (T) t position 24 in wild-type ALK to sprtic cid (D) using PCR with Pfu polymerse (Strtgene, L Joll, CA, USA) with primers contining the designed muttions, s descried previously. 39 N-terminl His-tgged kinse domin of constitutively ctive ALK (T24D) nd N-terminl His-tgged full-length Smd3 proteins were expressed in Sf9 insect cells using the BcNBlue culovirus expression system (Invitrogen, Crlsd, CA, USA). Proteins were purified with Ni-NTA resin column (Qigen, Vlenci, CA, USA). Purified Smd3 protein (2 ng) ws mixed with 1 ml of.1 M sodium icronte coting uffer nd plted onto Flsh-Pltes (Perkin Elmer, Wellesley, MA, USA). Pltes were covered nd incuted t 41C for 16 h. Pltes were llowed to lock in 1% ovine serum lumin in phosphte-uffered sline t room temperture for 1 h. Purified ALK protein (2 ng) in 2 ml of rection uffer (2 mm Tris-HCl, ph 7.4, mm MgCl 2,1mMCCl 2, 1mM dithiothreitol, 1 mm ATP nd 2 mci g- 32 P-ATP) ws mixed with different concentrtions of IN-113. ALK rection mixture ws dded onto Smd3-coted pltes nd incuted t 31C for 3 h. After incution, the pltes were wshed three times with 2 ml of 1 mm sodium pyrophosphte solution. After ir drying, the rdioctivity ws counted using Pckrd TopCount (Pckrd instrument compny, Meriden, CT, USA). The ssys were run in duplicte nd repeted twice. IC vlue ws clculted with SigmPlot softwre using sigmoidl dose response curve (n ¼ 4). Animl nd experimentl design Six-week-old mle Sprgue Dwley rts were purchsed from Orient Bio Inc. (Seoul, Repulic of Kore). Rts weighing 18 2 g were prepred. After mid-dominl incision under nesthesi using n intrperitonel injection of ketmine (3 mg/kg) nd xylzine (9 mg/ kg), ws chieved y ligting the left ureter with 4- silk t two points nd cutting etween ligtures. The nimls tht underwent surgery were rndomly divided into three groups: plus vehicle (n ¼ 11), plus low dose IN-113 (1 mg/kg, n ¼ 11), nd plus high dose IN-113 (2 mg/kg, n ¼ 11). -operted nimls (n ¼ 11) were used s control. Tretment strted within 1 h fter surgicl procedure. IN-113 ws prepred s descried previously. 26 Animls were intrperitonelly treted once dily with either vehicle (sline) or IN-113 for 7 (for ech group, n ¼ 7) or 14 (for ech group, n ¼ 4) dys. Animls were mintined in temperturecontrolled room (t 241C) nd supplied with utoclved food nd wter d liitum. At 7 nd 14 dys post-surgery, nimls were killed, nd the left kidneys were removed. The kidneys were sgittlly sliced into severl prts, snp frozen in liquid nitrogen nd kept t 81C. A prt of kidneys ws immersed into 1% neutrl-uffered formlin for histopthologicl nd immunohistochemicl exmintions. All experimentl procedures were conducted in ccordnce with our institutionl guidelines. Quntittive rel-time PCR nlyses Totl RNA ws isolted using Trizol regent (Invitrogen) ccording to the mnufcturer s instruction. Reverse trnscription ws performed using the ultr high-frequency reverse trnscriptse- PCR system kit (Strtegene). Synthesized complementry DNA ws mplified y stndrd PCR protocol using iq TM SYBR s Green supermix (Bio-Rd, Hercules, CA, USA) nd rt-specific primers for type 1 collgen (forwrd: -GGGACTTACCTCACTGCTTTTC-3 ; reverse: -AATCACGACGCTGGGACTG-3 ) nd for TGF-1 (forwrd: -CGGACTACTACGCCACCGCCGT-3 ; reverse: -TGGTTT TGTCATAGATTGCGTT-3 ). Prllel mplifictions with primers for hypoxnthine phosphoriosyltrnsferse (HPRT) (forwrd: -GGGACTTACCTCACTGCTTTTC-3 ; reverse: -AATCACGACG CTGGGACTG-3 ) were performed. Cycling conditions were: 3 min preincution t 91C, 1 s denturtion t 91C, 3 s nneling t 81C for 4 cycles using icycler (Bio-Rd). The fluorescent product ws detected t the end of ech cycle. Product specificity ws confirmed y grose gel electrophoresis nd routinely y meltingcurve nlysis. Rel-time PCR dt were nlyzed y using icycler softwre (Bio-Rd). The rtios of type 1 collgen nd TGF-1 to HPRT mrnas were clculted in ech smple. Additionlly, synthesized complementry DNA s descried ove ws mplified y stndrd PCR protocol using Tq polymerse nd rt-specific primers for TGF-1 nd for type I collgen. Prllel mplifictions with primers for HPRT were performed. The numers of cycles (etween 21 nd 24) giving the liner rnge nd nneling tempertures (etween 8 nd 61C) were djusted depending on the genes mplified. Rection products were electrophoresed on 2.% grose gels. Western lotting nlyses Kidney tissue lystes were prepred y homogeniztion of frozen tissues in rdioimmunoprecipittion ssy uffer contining,.% sodium deoxycholte, 1 mg/ml phenylmethnesulfonyl fluoride, 1% protese inhiitor cocktil, nd 1% phosphtse inhiitor cocktil (Sigm, St Louis, MO, USA). The lystes were clered y centrifugtion t 13 g t 41C for 2 min. The protein content of the lystes ws determined using the icinchoninic cid protein ssy kit (Pierce, Rockford, IL, USA). Proteins (2 mg) were seprted y 6 1% sodium dodecyl sulfte-polycrylmide gel electrophoresis, nd then trnsferred to nitrocellulose memrnes (Amershm Biosciences, Pisctwy, NJ, USA). The memrnes were proed with pproprite ntiodies in locking uffer including % ovine serum lumin or % nonft milk overnight t 41C. The primry ntiodies were rit ntiphospho-smd2 (Cell Signling Technology, Beverly, MA, USA), mouse nti--sma (Sigm), mouse nti-fironectin (BD Biosciences, Sn Jose, CA, USA), nd mouse nti--ctin (Sigm). The lots were incuted with pproprite secondry immunogloulin G-peroxidse conjugtes (1:1, dilution; Kirkegrd Kidney Interntionl (26) 7,

9 o r i g i n l r t i c l e J-A Moon et l.: IN-113 suppresses renl firosis Perry Lortories, Githersurg, MD, USA), nd developed using the enhnced chemiluminescence method. Bnd intensities were nlyzed using densitometer (Model GS-69; Bio-Rd) equipped with the Multi-Anlyst softwre progrm (Version 1.1; Bio-Rd). For quntifiction, the densities of the signls were normlized to tht for -ctin. Determintion of kidney hydroxyproline content Kidneys were completely hydrolyzed in 6 M HCl t 111C for 2 h. The otined dried pellets were reconstituted with citrte-cette uffer (ph 6.) contining.2 M citric cid,.2 M glcil cetic cid,.4 M sodium cette, nd.8 M sodium hydroxide. Smples were oxidized y 1 ml of chlormines-t solution (.282 g chlormines-t dded to 16 ml citrte-cette uffer, 2. ml of n-propnol, nd 2. ml H 2 O). After incuting the mixture for 2 min t room temperture, 1 ml of Ehrlich s solution (2. g of p-dimethylminoenzldehyde dded to 9.3 ml of n-propnol nd 3.9 ml of 7% perchloric cid) ws dded, nd the smples were incuted for 3 min t 61C. The sornce of ech smple ws mesured t 7 nm using spectrophotometer. Hydroxyproline content ws clculted from the stndrd curve constructed with hydroxy-lproline (Sigm). The results were expressed s hydroxyproline per mg protein. Protein ws determined using the icinchoninic cid protein ssy kit (Pierce). Histopthologicl exmintion Fixed tissues in 1% neutrl-uffered formlin were dehydrted in grded lcohol nd emedded in prffin. Prffin sections ( mm thick) were stined with hemtoxylin eosin or with Msson smodified trichrome. Immunohistochemicl exmintion For immunohistochemicl nlysis, prffin-emedded sections ( mm) were deprffinized, wshed with phosphte-uffered sline nd treted with 3% H 2 O 2 for min. An ntigen retrievl protocol in which sections were heted in 1 mm citrte uffer (ph 6.) for 1 min ws used for psmd2 immunohistochemistry. Sections were locked with 1% norml got serum in Tris-HCl-uffered sline or horse serum in phosphte-uffered sline for 1 h nd then incuted with either nti-psmd2 (1:2) ntiody t 41C overnight or with -SMA (1:2) ntiody diluted in locking solution for 1 h t room temperture. After wshing, sections were incuted with pproprite iotin-conjugted secondry immunogloulin G, nd then treted with regents from Vect-Elite streptvidin-peroxidse kit (Vector Lortories, Burlingme, CA, USA) with enzidine sustrte for color development. Sections were counterstined with diluted hemtoxylin. Sttisticl nlyses Quntittion of Western lotting dt ws performed y mesuring the intensity of the signls using Quntity One Imge nlysis softwre (Bio-Rd). Dt re expressed s s.e.m. Differences etween groups were exmined for sttisticl significnce y using nlysis of vrince. Sttisticl nlysis of the dt ws performed further y using lest significnt difference test etween the two groups. Po. were considered to e significnt. ACKNOWLEDGMENTS This work ws supported y the grnts from Ministry of Commerce, Industry nd Energy, Kore (M nd M ). REFERENCES 1. Heldin CH, Miyzono K, ten Dijke P. TGF-et signlling from cell memrne to nucleus through SMAD proteins. Nture 1997; 39: Mssgue J. TGF-et signl trnsduction. Annu Rev Biochem 1998; 67: Lesk A, Arhm DJ. TGF-et signling nd the firotic response. FASEB J 24; 17: Wng W, Kok V, Ln HY. Trnsforming growth fctor-et nd Smd signlling in kidney diseses. Nephrology (Crlton) 2; 1: ten Dijke P, Hill CS. New insights into TGF-et-Smd signling. Trends Biochem Sci 24; 29: Agrotis A, Klinin N, Boik A. Trnsforming growth fctor-et, cell signling nd crdiovsculr disorders. Curr Vsc Phrmcol 2; 3: Elliott RL, Bloe GC. Role of trnsforming growth fctor Bet in humn cncer. J Clin Oncol 2; 23: Bello-DeOcmpo D, Tindll DJ. 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