Effects of colchicine on renal fibrosis and apoptosis in obstructed kidneys

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1 ORIGINAL ARTICLE Koren J Intern Med 8;: Effects of colchicine on renl firosis nd poptosis in ostructed kidneys Sejoong Kim,, Eun Sook Jung, Jeonghwn Lee, Nm Ju Heo,, Ki Young N,, nd Jin Suk Hn Deprtment of Internl Medicine, Seoul Ntionl University College of Medicine, Seoul; Deprtment of Internl Medicine, Seoul Ntionl University Bundng Hospitl, Seongnm; Deprtment of Internl Medicine, Hllym University Hngng Scred Hert Hospitl, Seoul; Deprtment of Internl Medicine, Seoul Ntionl University Hospitl Helthcre System Gngnm Center, Seoul, Kore Received : April 8, 6 Revised : July, 6 Accepted : July 6, 6 Correspondence to Jin Suk Hn, M.D. Deprtment of Internl Medicine, Seoul Ntionl University Hospitl, Dehk-ro, Jongno-gu, Seoul 8, Kore Tel: Fx: E-mil: jshn@snu.c.kr Bckground/Aims: Colchicine is n estlished drug for microtuule stiliztion tht my reduce tissue injury. No dt were ville tht its effects my depend on the dosge of colchicine. We investigted the nti-firotic nd poptotic effects of vrious dose of colchicine in unilterl ureterl ostruction (UUO) model. Methods: Thirty-six Sprgue-Dwley rts were rndomly ssigned into six groups. Two shm groups were divided into vehicle-treted or colchicine-treted group ( μg/kg/dy). Four UUO groups were treted with either vehicle or three different doses of colchicine for 7 dys (, 6, nd μg/kg/dy, intrperitonelly). All of the nimls were scrificed on dy 7. Results: Colchicine tretment diminished cetylted α-tuulin nd tumor growth fctor-β immunorectivities in the corticl re of the 7-dy ostructed kidneys, which ws in dose dependent mnner. Colchicine ttenuted tuulointerstitil dmge nd poptosis in oth corticl nd medullry re, nd eneficil effects of colchicine therpy were drmticlly shown t the higher dosge of colchicine. The expression levels of cleved cspse-, ED-, nd fironectin were decresed in UUO nimls. Conclusions: We found tht the proper dosge of colchicine my hve nti-firotic nd nti-poptotic effects in ostructed kidneys. For clinicl pplictions, n optiml dose of colchicine should e evluted to mximize the prevention of renl disese progression. Keywords: Colchicine; Kidney; Microtuules; Ureterl ostruction INTRODUCTION As the numer ptients with chronic kidney disese rpidly grows, need for novel nd effective mediction for renl disese is rising. Discovering unknown effects of estlished drugs will e nother wy to meliorte the progression of renl disese. Colchicine is microtuule-trgeting gent which depolymerizes microtuules nd locks mitosis []. Colchicine is used s n nti-cncer mediction for poptosis induction in cncer cells. Additionlly, it hs previously een used to relieve cute gout flres. In contrst, some reserchers hve demonstrted tht microtuule ltertion using colchicine induces survivl signls []. Colchicine hs demonstrted its nti-firotic nd nti-inflmmtory effects in severl kidney models of crescentic nephritis [], chronic cyclosporine nephrotoxicity [], dietic nephropthy [5], nd hypertensive chronic kidney disese [6]; however, these studies used only single dosge of colchicine. The kidney hs functionl nephron heterogeneity, suggesting tht the medull is much more susceptile thn the cortex to ostructive nephropthy nd renl ischemi [7,8]. However, the mjority of the previous studies did not ddress this dispr- Copyright 8 The Koren Assocition of Internl Medicine This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( y-nc/./) which permits unrestricted noncommercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. pissn 6- eissn

2 Kim S, et l. Colchicine nd renl firosis ity etween the cortex nd medull nor did they evlute these res seprtely. Unilterl ureterl ostruction (UUO) is good model of tuulr poptosis nd interstitil firosis in the kidneys [9,]. Mechnicl stress induces the over-polymeriztion of microtuules, nd contriutes to tissue morphogenesis, remodeling, nd firosis []. We investigted the microtuule-depolymerizing effects of vrious doses of colchicine on renl firosis nd poptosis t oth the surfce nd deep nephron during ostructive nephropthy. Our findings provide new perspective on the renoprotective effects of colchicine. METHODS The Institutionl Animl Cre nd Use Committee (Seoul Ntionl University Bundng Hospitl) pproved the experimentl protocol (No. BA-9/7-). Thirty-six specific pthogen-free mle Sprgue-Dwley rts (5 to 6 weeks old; Orient Bio Inc., Seongnm, Kore), initilly weighing pproximtely g, were kept in temperture-controlled room nd were llowed free ccess to stndrd lortory chow nd tp wter. UUO ws performed y ligtion of the left ureter nd shm opertion ws identicl to the UUO opertion except for the ligtion of the ureter. A totl of rts comprised the shm groups (n = ) nd the UUO groups (n = ). The shm groups were divided into vehicle-treted group nd colchicine-treted group ( µg/kg/dy); the UUO groups were divided in dose dependent mnner (vehicle,, 6, nd µg/kg/dy colchicine tretment). For 7 dys, the rts in the vehicle group received dily n intrperitonel injection of only sterile sline, nd the rts in the colchicine group received colchicine (Sigm-Aldrich Corp., Yongin, Kore) intrperitonelly on dily sis for 7 dys. On dy 7, ll of the nimls were scrificed, nd the kidneys were isolted nd immeditely stored t 7 C until use. Blocks of kidney were fixed in % uffered formlin nd emedded in prffin for routine histologicl exmintion. The glomerulosclerosis index ws ssessed on periodic-cid Schiff-stined prffin sections t mgnifiction of. The tuulointerstitil injury indexes were determined using semiquntittive scoring system on Msson s trichrome-stined sections t mgnifiction of. More thn consecutive fields were exmined nd the grding ws performed in lind mnner. The tuulointerstitil injury score ws estimted sed on the tuulr trophy, interstitil inflmmtion, firosis nd csts in the rnge of to (, no chnge;, < 5%;, 5% to 5%; nd, 5% to %) []. Apoptosis ws detected y enzymticlly leling DNA strnd reks using terminl deoxynucleotidyl trnsferse dutp nick end leling (TUNEL) stining. TUNEL stining ws performed using Cell Deth Detection Kit (Roche, Mnnheim, Germny). To revel the totl quntity of nuclei, the sme slides were stined with,6 -dimidino--phenylindole nd the TUNEL-positive cells were counted in t lest different fields under mgnifiction. Positive stining ws detected using lser-scnning confocl microscopy (Zeiss LSM 7, Crl Zeiss, Oerkochen, Germny). The kidneys were dissected into the cortex nd inner stripe of the outer medull nd ech region ws seprtely homogenized using tissue homogenizer (PowerGun 5, Fisher Scientific, Pittsurgh, PA, USA) []. Ech protein nlyzed using Pierce BCA Protein Assy Kit (Pierce Biotechnology, Rockford, IL, USA). The smples were frctionted y SDS-PAGE (sodium dodecyl sulfte polycrylmide gel electrophoresis). After trnsfer onto the protrn nitrocellulose trnsfer memrne (Whtmn, Gssel, Germny), the lots were proed using the following ntiodies: monoclonl mouse nti-cetylted α-tuulin ntiody (Acm, Cmridge, UK) nd nti-α-tuulin (Snt Cruz Biotechnology, Snt Cruz, CA, USA), nti-vimentin (Snt Cruz Biotechnology), nti-e-cdherin (BD iosciences, Sn Jose, CA, USA), nti-α-smooth muscle ctin (Acm), nti-fironectin (F68, Sigm-Aldrich, St. Louis, MO, USA), nti-cspse- (Cell Signling Technology, Dnvers, MA, USA), nti-cspse-9 (BD Biosciences), ED- (MCA957, AD serotec, Oxford, UK), nd got polyclonl nti-β-ctin (Snt Cruz Biotechnology). Incution with peroxidse-conjugted nti-rit or nti-got IgG secondry ntiodies (Pierce Biotechnology) ws followed y nd visuliztion using enhnced chemiluminescence (ECL RPN 6, Amershm Phrmci Biotech, Buckinghmshire, UK). The nd densities were quntified y densitometry (GS-7 Imging Densitometry, Bio-Rd, Hercules, CA, USA). Tumor growth fctor β (TGF-β) immunossy system were purchsed from Promeg 569

3 The Koren Journl of Internl Medicine Vol., No., My 8 Rtio of controls (cortex) kd 7 kd Acetylted α-tuulin α-tuulin TGF-β Rtio of controls (cortex)..5. SC UV UC UC6 UC 55 kd 7 kd TGF-β/protein (pg/mg) cortex 6 6 Rtio of controls (medull)..5. SC UV UC UC6 UC Rtio of controls (medull)..5. TGF-β/protein (pg/mg) medull 55 kd 55 kd 7 kd 7 kd A B C Figure. Comprison of (A) cetylted α-tuulin, (B) α-tuulin, nd (C) tumor growth fctor β (TGF-β) using colchicine tretment in ureterl ostructive kidneys. The expression of cetylted α-tuulin ws incresed in unilterl ureterl ostruction (UUO)-operted rts nd ws diminished in the corticl lesions of colchicine-treted UUO rts. The corticl expression of TGF-β ws incresed fter UUO, nd then grdully decresed during colchicine tretment., shm-operted rts with vehicle; SC, shm-operted rts with colchicine tretment; UV, ureterl ostructive rts with vehicle; UC, ureterl ostructive rts with colchicine tretment. p <.5, p <.. (Mison, WI, USA) Sttisticl nlysis Comprisons etween two groups were mde with the Mnn-Whitney U test, nd comprisons mong three groups were mde with the Kruskl-Wllis test (SPSS version 8., SPSS Inc., Chicgo, IL, USA). Bnd density vlues were stndrdized y dividing them y the verge vlue of the control group. The men for the control group ws defined s %, nd the results were expressed s the men ± SEM. A p <.5 ws considered sttisticlly significnt. RESULTS To evlute the effects of colchicine on microtuule polymeriztion, immunolots of cetylted α-tuulin, α-tuulin, nd vimentin were performed. The immunolotting of cetylted α-tuulin in UUO-operted rts ws slightly incresed compred to the shm-operted rts nd ws diminished in the corticl lesion of the 6- nd -mg colchicine-treted UUO rts (Fig. ). The immunorectivity of α-tuulin ws not significntly chnged in colchicine- or vehicle-treted UUO rts. Although immunolotting demonstrted tht vimentin ws lso incresed in the UUO models, colchicine tretment filed to exhiit differences in the UUO rts (dt not shown). The corticl expres- 57

4 Kim S, et l. Colchicine nd renl firosis Glomerulosclerosis index PAS..5 MT (Cx). MT (Med) Tuulointerstitil dmge incex (Cx) Tunel (Cx) Tunel (Med) Tuulointerstitil dmge incex (Med) SC UV UC UC6 UC Apoptosis cell (%) (Cx) 5 5 sion of TGF-β ws lso incresed fter UUO, nd ws grdully decresed under colchicine tretment (Fig. ). The incresed dosge of colchicine tretment in UUO nimls leded to decresed expression of cetylted α-tuulin nd TGF-β, compred to the vehicle-treted UUO nimls. We oserved glomerulosclerosis, tuulointerstitil firosis, nd tuulr poptosis s the primry endpoints of this niml study. The glomerulosclerosis indices were the sme mong ll six groups determined y peri- 5 Apoptosis cell (%) (Med) Figure. Pthologic findings in ureterl ostructive kidneys: glomerulosclerosis (periodic-cid Schiff [PAS] stin, ), tuulointerstitil dmge (Msson s-trichrome [MT] stin, ), nd poptosis (terminl deoxynucleotidyl trnsferse dutp nick end leling [TUNEL] stin, ). Tuulointerstitil dmge nd poptosis were ttenuted fter colchicine tretment. Cx, corticl lesion; Med, medullry lesion;, shm-operted rts with vehicle; SC, shm-operted rts with colchicine tretment; UV, ureterl ostructive rts with vehicle; UC, ureterl ostructive rts with colchicine tretment. p <.5, p <.. 5 odic-cid Schiff stining. The tuulr dmge nd interstitil firosis scores s reveled y Msson s trichrome stining were incresed in the UUO rts compred to the shm-operted rts, nd these scores in oth corticl nd medullry lesions were ttenuted in the high-dose colchicine-treted UUO rts to greter degree compred to vehicle-treted UUO rts (Fig. ). The numer of tuulr poptotic cells ws significntly incresed fter the UUO opertion, ut ws significntly reduced in oth corticl nd medullry lesions with colchicine 57

5 The Koren Journl of Internl Medicine Vol., No., My 8 Fironectin E-cdherin α-smooth muscle ctin Rtio of controls (cortex) Rtio of controls (cortex)...5 Rtio of controls (cortex) SC UV UC UC6 UC.. kd kd kd 7 kd 7 kd 7 kd Rtio of controls (medull) 8 6 Rtio of controls (medull)..5 Rtio of controls (medull) 5 5 SC UV UC UC6 UC. kd 7 kd kd 7 kd kd 7 kd A B C Figure. Immunolots relted to firosis: (A) fironectin, (B) E-cdherin, nd (C) α-smooth muscle ctin. Fironectin expression ws reduced with colchicine therpy; epithelil-mesenchyml trnsition mrkers were not chnged., shm-operted rts with vehicle; SC, shm-operted rts with colchicine tretment; UV, ureterl ostructive rts with vehicle; UC, ureterl ostructive rts with colchicine tretment. p <.5, p <.. tretment in dose-dependent mnner (Fig. ). Fironectin ws mrkedly incresed in the UUO models, wheres it ws significntly meliorted with colchicine tretment in the medullry lesions of UUO rts (Fig. ). The expression of E-cdherin decresed while α-smooth muscle ctin incresed fter ureterl ostruction; however, there were no differences etween the colchicine- nd vehicle-treted groups. The expression of poptosis-relted protein, including cleved cspse- nd cspse-9, ws exmined. Cleved cspse- ws incresed in UUO models nd ws rogted in corticl nd medullry lesions with incresing doses of colchicine (Fig. ). The expression of cspse-9 ws slightly decresed with colchicine tretment in the UUO rts, ut no significnt differences were found (Fig. ). Immunolots of ED- were lso evluted to quntify inflmmtion. In the UUO rts, the expression of ED- ws incresed compred to the shm-operted controls, wheres ED- expression ws ttenuted fter colchicine tretment in the UUO rts, especilly in medullry lesions (Fig. ). 57

6 Kim S, et l. Colchicine nd renl firosis Cleved cspse Cspse 9 ED-..5 Rtio of controls (cortex) Rtio of controls (cortex)..5 Rtio of controls (cortex)...5 SC UV UC UC6 UC.. 9 kd 7 kd 7 kd 7 kd kd 7 kd 7 kd 7 kd Rtio of controls (medull) Rtio of controls (medull) Rtio of controls (medull) 5. 9 kd 7 kd 7 kd 7 kd 7 kd 7 kd A B C Figure. Immunolots relted to poptosis nd inflmmtion: (A) cleved cspse-, (B) cspse-9, nd (C) ED-. The cspse pthwy ws locked nd ED- ws slightly decresed with high-dose colchicine therpy., shm-operted rts with vehicle; SC, shm-operted rts with colchicine tretment; UV, ureterl ostructive rts with vehicle; UC, ureterl ostructive rts with colchicine tretment. p <.5, p <.. kd 7 kd DISCUSSION We found tht n pproprite dosge of colchicine cn meliorte renl firosis nd poptosis in ostructive nephropthy, indicting tht colchicine cn e used to prevent the progression of kidney injury. The depolymerizing effect of colchicine my led to the ttenution of renl firosis nd poptosis in ostructed kidneys. We found tht ureterl ostruction my induce the over-ctivtion nd ltertion of microtuules in corticl lesions. Exogenous mechnicl forces contriute to the overexpression of cetylted tuulin []. Colchicine is known to inhiit the polymeriztion of tuulin [] nd the cetyltion of tuulin polymers [5]. When we evluted cytoskeletl components such s microtuules (cetylted α-tuulin nd α-tuulin) nd intermedite filments (vimentin), we confirmed the decresed expression of cetylted α-tuulin fter tretment with colchicine in the UUO kidneys. TGF-β plys crucil role in the promotion of renl firosis, nd colchicine suppresses TGF-β expression nd diminishes cyclosporine relted nephrotoxicity [6,7], which my e con- 57

7 The Koren Journl of Internl Medicine Vol., No., My 8 sistent with our results tht colchicine tretment led to down-regultion of TGF-β nd reduced renl firosis. Some reserchers hve reported tht the decetyltion of α-tuulin y histone decetylse 6 contriutes to the TGF-β relted epithelil mesenchyml trnsition [8]. However, this is inconsistent with our finding tht the ttenution of cetylted α-tuulin y colchicine ws ssocited with decresed TGF-β expression. Acetylted α-tuulin is present t the junction etween T-cell nd n ntigen presenting cell, nd the decetyltion of tuulin my ttenute the lymphocyte ctivtion signling cscde [9]. This is consistent with our finding tht ED-, cellulr mrker tht is specific for mcrophges, hs een shown to e decresed y colchicine tretment in UUO models. It is possile tht the ttenution of inflmmtory cells my led to reduced TGF-β production, which my e ssocited with the nti-poptotic nd nti-firotic effects of colchicine tretment. We found tht high dose colchicine led to reduction in cetylted α-tuulin nd cspse- expression in the corticl lesions of UUO rts. This is consistent with previous report tht found tht the cetyltion of α-tuulin led to the ctivtion of cspse- [], which my e relted to renl poptosis []. In the current study, only cspse- expression nd tuulr poptosis were ttenuted in medullry lesions without microtuule ltertions. The extrcellulr signl-regulted kinse (ERK) pthwy my e involved in mechnicl stress-induced hypertrophic responses []. The initil ctivtion of ERK fter UUO ws restricted to the medullry collecting ducts nd the thick scending lim of Henle, which precedes tuulr prolifertion nd injury []. In vitro studies showed tht colchicine cuses inctivtion of ERK []. It could e possile tht microtuule independent nti-poptotic effect of colchicine my occur in the medullry lesions of ostructed kidneys. Some studies demonstrted n pprent interply etween erly poptosis nd susequent firosis, indicting tht poptosis my e n erly event tht precedes the onset of firosis []. Although we filed to show tht colchicine prevents the epithelil mesenchyml trnsition, colchicine-relted nti-poptotic effect my meliorte kidney firosis. We showed tht fironectin expression ws significntly meliorted with colchicine tretment in medullry lesions. This finding my e consistent with dt tht show the effect of colchicine on collgen ccumultion y inhiiting fironectin trnsltion nd expression [6,]. Fironectin expression ws decresed fter colchicine tretment in UUO kidneys, which is consistent with previous reports [5]. In contrst, α-smooth muscle ctin expression ws not chnged fter colchicine tretment. α-smooth muscle ctin is normlly expressed in the smooth muscle lineges cells, prticulrly firolsts. Fironectin is glycoprotein of the extrcellulr mtrix tht is secreted y firolsts. This suggests colchicine tretment my inhiit the secretory function of firolsts, ut my not completely inhiit the prolifertion of firolsts in this UUO model. This study hs some limittions. We did not mesure renl function using prmeters such s serum cretinine levels or urinry iomrkers. However, tissue injury indices re used in mny UUO models s outcomes of tretment [5,6]. It is possile tht colchicine my retrd the exocytosis of intrcellulr vesicles such s those expressing quporin- nd H+-ATPse vi microtuule ltertions tht could e relted to impired urine concentrtion nd tuulr cidifiction [7,8]. Unfortuntely, we did not evlute the expression of those vesicles; this should e evluted in the future. In ddition, longer tretment with colchicine should e evluted, s these dt were limited to 7-dy UUO models. We found tht colchicine my hve eneficil effects on renl poptosis nd firosis in ostructed kidneys. For clinicl pplictions, n pproprite dose of colchicine should e evluted to mximize the prevention of renl disese progression. KEY MESSAGE. Colchicine hs nti-firotic nd nti-poptotic effects in the ostructed kidneys.. Those eneficil effects my depend on the dosge of colchicine.. For clinicl pplictions, n optiml dose of colchicine should e evluted to mximize the prevention of renl disese progression. Conflict of interest No potentil conflict of interest relevnt to this rticle ws reported. 57

8 Kim S, et l. Colchicine nd renl firosis Acknowledgments This work ws supported y grnt No. RAA689 from the Ntionl Reserch Foundtion of Kore, nd grnd of the Kore Helth Technology R&D Project through the Kore Helth Industry Development Institute (KHIDI), funded y the Ministry of Helth nd Welfre, Repulic of Kore (grnt numer: HI6C). REFERENCES. Kim SK, Cho SM, Kim H, et l. The colchicine derivtive CT6 shows novel microtuule-modulting ctivity with poptosis. Exp Mol Med ;5:e9.. Mollinedo F. Survivl nd poptotic signls in the ction of microtuule-trgeting ntitumor drugs. IDrugs 5;8:7-.. McClurkin C Jr, Phn SH, Hsu CH, et l. Moderte protection of renl function nd reduction of firosis y colchicine in model of nti-gbm disese in the rit. J Am Soc Nephrol 99;: Li C, Yng CW, Ahn HJ, et l. Colchicine decreses poptotic cell deth in chronic cyclosporine nephrotoxicity. J L Clin Med ;9: Li JJ, Lee SH, Kim DK, et l. Colchicine ttenutes inflmmtory cell infiltrtion nd extrcellulr mtrix ccumultion in dietic nephropthy. Am J Physiol Renl Physiol 9;97:F-F9. 6. Gun T, Go B, Chen G, et l. Colchicine ttenutes renl injury in model of hypertensive chronic kidney disese. Am J Physiol Renl Physiol ;5:F66-F Wilson DR. Nephron functionl heterogeneity in the postostructive kidney. Kidney Int 975;7: Mson J, Welsch J, Tktke T. Disprity etween surfce nd deep nephron function erly fter renl ischemi. Kidney Int 98;: Goe GC, Axelsen RA. Genesis of renl tuulr trophy in experimentl hydronephrosis in the rt. Role of poptosis. L Invest 987;56:7-8.. Truong LD, Choi YJ, Tso CC, et l. Renl cell poptosis in chronic ostructive uropthy: the roles of cspses. Kidney Int ;6:9-9.. Yuto X, Geru W, Xiojun B, To G, Aiqun M. Mechnicl stretch-induced hypertrophy of neontl rt ventriculr myocytes is medited y et()-integrin-microtuule signling pthwys. Eur J Hert Fil 6;8:6-.. Kim S, Heo NJ, Jung JY, et l. Chnges in the sodium nd potssium trnsporters in the course of chronic renl filure. Nephron Physiol ;5:p-p.. N KY, Kim GH, Joo KW, et l. Chronic furosemide or hydrochlorothizide dministrtion increses H+-ATPse B suunit undnce in rt kidney. Am J Physiol Renl Physiol 7;9:F7-F79.. Gourly CW, Ayscough KR. The ctin cytoskeleton: key regultor of poptosis nd geing? Nt Rev Mol Cell Biol 5;6: Jung HI, Shin I, Prk YM, Kng KW, H KS. Colchicine ctivtes ctin polymeriztion y microtuule depolymeriztion. Mol Cells 997;7: Disel U, Pyds S, Dogn A, Gulfiliz G, Yvuz S. Effect of colchicine on cyclosporine nephrotoxicity, reduction of TGF-et overexpression, poptosis, nd oxidtive dmge: n experimentl niml study. Trnsplnt Proc ;6: El-Skk AI, Bkircioglu ME, Bhtngr RS, Yen TS, Dhiy R, Lue TF. The effects of colchicine on Peyronie s-like condition in n niml model. J Urol 999;6: Shn B, Yo TP, Nguyen HT, et l. Requirement of HDAC6 for trnsforming growth fctor-et-induced epithelil-mesenchyml trnsition. J Biol Chem 8;8: Serrdor JM, Crero JR, Sncho D, Mittelrunn M, Urzinqui A, Snchez-Mdrid F. HDAC6 decetylse ctivity links the tuulin cytoskeleton with immune synpse orgniztion. Immunity ;:7-8.. Ruwhof C, vn der Lrse A. Mechnicl stress-induced crdic hypertrophy: mechnisms nd signl trnsduction pthwys. Crdiovsc Res ;7:-7.. Stone AA, Chmers TC. Microtuule inhiitors elicit differentil effects on MAP kinse (JNK, ERK, nd p8) signling pthwys in humn KB- crcinom cells. Exp Cell Res ;5:-9.. Mski T, Foti R, Hill PA, Ikezumi Y, Atkins RC, Nikolic-Pterson DJ. Activtion of the ERK pthwy precedes tuulr prolifertion in the ostructed rt kidney. Kidney Int ;6: To Y, Kim J, Fuel S, et l. Cspse inhiition reduces tuulr poptosis nd prolifertion nd slows disese progression in polycystic kidney disese. Proc Ntl Acd Sci U S A 5;: Zhou B, Rinovitch M. Microtuule involvement in 575

9 The Koren Journl of Internl Medicine Vol., No., My 8 trnsltionl regultion of fironectin expression y light chin of microtuule-ssocited protein in vsculr smooth muscle cells. Circ Res 998;8: Prk SH, Choi MJ, Song IK, et l. Erythropoietin decreses renl firosis in mice with ureterl ostruction: role of inhiiting TGF-et-induced epithelil-to-mesenchyml trnsition. J Am Soc Nephrol 7;8: Chevlier RL, Fores MS, Thornhill BA. Ureterl ostruction s model of renl interstitil firosis nd ostructive nephropthy. Kidney Int 9;75: Kwon TH, Frokier J, Nielsen S. Regultion of quporin- in the kidney: moleculr mechnism of ody-wter homeostsis. Kidney Res Clin Prct ;: Kng SH, Kim J, Prk JW. Biopsy-proven type renl tuulr cidosis in ptient with metolic cidosis. Koren J Intern Med ;7: