The intrinsic prostaglandin E 2 EP 4 system of the renal tubular epithelium limits the development of tubulointerstitial fibrosis in mice

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1 originl rticle & 1 Interntionl Society of Nephrology see commentry on pge 13 The intrinsic prostglndin E EP system of the renl tuulr epithelium limits the development of tuulointerstitil firosis in mice Noki Nkgw 1,, Koh-ichi Yuhki 1,3, Jun-ichi Kwe, Tkyuki Fujino 1,, Osmu Tkht 1, Mki Kr, Kzutoshi Ae, Fumiki Kojim 1,3, Hitoshi Kshiwgi 1,3, Noyuki Hsee, Kenjiro Kikuchi, Yukihiko Sugimoto, Shuh Nrumiy 3,5 nd Fumitk Ushikui 1,3 1 Deprtment of Phrmcology, Ashikw Medicl University, Ashikw, Jpn; First Deprtment of Internl Medicine, Ashikw Medicl University, Ashikw, Jpn; 3 CREST, Jpn Science nd Technology Agency, Tokyo, Jpn; Deprtment of Physiologicl Chemistry, Grdute School of Phrmceuticl Sciences, Kyoto University, Kyoto, Jpn nd 5 Deprtment of Phrmcology, Kyoto University Fculty of Medicine, Kyoto, Jpn Inflmmtory responses in the kidney led to tuulointerstitil firosis, common feture of chronic kidney diseses. Here we exmined the role of prostglndin E (PGE ) in the development of tuulointerstitil firosis. In the kidneys of wild-type mice, unilterl ureterl ostruction leds to progressive tuulointerstitil firosis with mcrophge infiltrtion nd myofirolst prolifertion. This ws ccompnied y n upregultion of COX- nd PGE receptor sutype EP mrnas. In the kidneys of EP gene knockout mice, however, ostruction-induced histologicl ltertions were significntly ugmented. In contrst, n EP - specific gonist significntly ttenuted these ltertions in the kidneys of wild-type mice. The mrnas for mcrophge chemokines nd profirotic growth fctors were upregulted in the kidneys of wild-type mice fter ureterl ostruction. This ws significntly ugmented in the kidneys of EP - knockout mice nd suppressed y the EP gonist ut only in the kidneys of wild-type mice. Notly, COX- nd MCP-1 proteins, s well s EP mrna, were loclized in renl tuulr epithelil cells fter ureterl ostruction. In cultured renl firolsts, nother EP -specific gonist significntly inhiited PDGF-induced prolifertion nd profirotic connective tissue growth fctor production. Hence, n endogenous PGE EP system in the tuulr epithelium limits the development of tuulointerstitil firosis y suppressing inflmmtory responses. Kidney Interntionl (1) 8, ; doi:1.138/ki.1.115; pulished online 18 April 1 KEYWORDS: chronic kidney disese; prostglndins; renl firosis Correspondence: Fumitk Ushikui, Deprtment of Phrmcology, Ashikw Medicl University, Midorigok Higshi , Ashikw , Jpn. E-mil: ushikui@shikw-med.c.jp Received 9 Novemer 1; revised 3 Jnury 1; ccepted 31 Jnury 1; pulished online 18 April 1 The worldwide increse in the numer of ptients with chronic kidney disese nd the resultnt end-stge renl filure necessitting renl replcement therpy, such s trnsplnttion nd dilysis, is thretening to rech lmost pndemic proportions. 1 Tuulointerstitil firosis, s well s glomerulosclerosis, is mjor predictor of the progression to end-stge renl filure in vrious types of chronic kidney disese. For exmple, interstitil firosis, long with glomerulosclerosis, is one of the most importnt fctors determining the progression nd prognosis of dietic nephropthy. 3 In immunogloulin A (IgA) nephropthy, which is the most common type of chronic glomerulonephritis chrcterized y glomerulr IgA1 deposition, tuulointerstitil firosis is more closely relted to prognosis of the disese thn to the glomerulr dmge itself. Therefore, clrifiction of the mechnism leding to tuulointerstitil firosis is n importnt issue for the mngement of chronic kidney disese. Tuulr epithelil cells re thought to ply pivotl role in the process of tuulointerstitil firosis. These cells produce severl types of chemokines in response to vrious stimuli nd ttrct mcrophges nd lymphocytes into the renl interstitil spce. 5 Among these chemokines, MCP-1 (monocyte chemottrctnt protein-1) nd RANTES (regulted on ctivtion, T cell expressed, nd secreted), which elong to the CC chemokine fmily, hve emerged s importnt meditors of mcrophge recruitment in vrious types of renl diseses. 6 8 The recruited mcrophges induce prolifertion of resident firolsts nd their differentition into myofirolsts through the production of profirotic growth fctors, such s trnsforming growth fctor-1 (TGF-1) nd connective tissue growth fctor (CTGF). 9,1 TGF-1 is lso known s the min plyer in the phenotypic conversion of tuulr epithelil cells into mesenchyml firolsts, process known s epithelil mesenchyml trnsition (EMT). 11 Therefore, tuulr epithelil cells, s well s resident firolsts nd pericytes, re le to serve s source of myofirolsts. 1, Kidney Interntionl (1) 8,

2 N Nkgw et l.: Prostglndin E in renl firosis originl rticle However, recently, Humphreys et l. 1 indicted no evidence of EMT in unilterl ureterl ostruction (UUO) model using novel fte-mpping technique, indicting some rguments out the role of EMT t lest in tuulointerstitil firosis induced y UUO. Differentited nd ctivted myofirolsts then produce interstitil mtrices nd collgen fier, resulting in tuulointerstitil firosis. Although these sequentil events tke plce in the development of tuulointerstitil firosis, the fctors prticipting in the regultion of these events hve not een fully chrcterized. UUO invrily induces tuulointerstitil firosis in the ureter-ostructed kidney (UOK), well-estlished model used to investigte the mechnism leding to tuulointerstitil firosis. 8,15,16 It hs een reported tht prostglndin E (PGE ) production increses significntly in the kidney during the development of tuulointerstitil firosis induced y UUO. 17,18 However, the role of PGE in the development of tuulointerstitil firosis remins to e determined. PGE exerts vriety of ctions in the ody y inding to its specific rhodopsin-type receptors. 19 There re four sutypes of PGE receptors encoded y different genes: EP 1,EP,EP 3,ndEP. EP 1 couples to G q nd rises intrcellulr C þ concentrtion. EP nd EP couple to G s nd rise intrcellulr camp concentrtion ([camp] i ). Notly, EP hs nother signling pthwy involving phosphtidylinositol 3-kinse, leding to the ctivtion of Akt nd extrcellulr signl regulted kinses.,1 In contrst, EP 3 couples minly to G i nd inhiits the increse in [camp] i. On the sis of the finding tht the expression levels of mrnas for cyclooxygense (COX)-, rte-limiting enzyme of prostnoid synthesis, nd EP were significntly upregulted in the kidney fter UUO, we set out to clrify the EP -medited role of PGE in the development of tuulointerstitil firosis using mice tht lck EP (EP mice) nd ONO-819, specific EP gonist. We found tht oth endogenous PGE nd ONO-819 significntly suppress UUO-induced tuulointerstitil firosis vi EP in vivo. In ddition, specific EP gonist ONO-AE1-39 significntly inhiited pltelet-derived growth fctor (PDGF)-BB-induced firolst prolifertion nd TGF-1-induced EMT in vitro. RESULTS Renl expression levels of COX- nd EP mrnas increse significntly in mice fter UUO In wild-type () mice, hydronephrotic chnges, including diltion of the renl pelvis nd thinning of the renl prenchym, occurred in UOK fter UUO (Figure 1). In ddition, these chnges were ccompnied y mrked collgen deposition in the renl interstitil re (Figure 1), indicting the development of tuulointerstitil firosis in the present UUO model. We investigted whether prostnoids nd their receptor system in the kidney respond to stress urdened y UUO in mice. First, we exmined the expressions of mrnas for COX-1 nd COX-. The expression level of mrna for COX-, n inducile isoform of COX, incresed significntly in UOK c mrna/18s d 1 mrna/18s CLK UOK CLK UOK COX-1 Control CLK UOK d1 Control UOK d1 UOK d UOK d7 d d7 COX- EP 1 EP EP 3 EP Figure 1 A hydronephrotic chnge with interstitil firosis nd expression of cyclooxygense (COX) nd prostglndin E receptor (EP) mrnas in wild-type () kidneys fter unilterl ureterl ostruction (UUO). () A representtive photogrph showing gross ppernce of the kidneys on dy 7 of UUO. The ureter-ostructed kidney (UOK) exhiits hydronephrotic chnge with dilted pelvis. () Sirius red stined coronl sections of the kidneys on dy 7 of UUO. In the section of UOK, collgen fiers stined red re evident. (c) Expression levels of COX-1 nd COX- mrnas in the kidney on dy 1 (d1), dy (d), nd dy 7 (d7) of UUO. Po.5 vs. respective control. w Po.5 vs. respective contrlterl kidney (CLK); n ¼ 5. (d) Expression levels of EP mrnas in UOK on d1, d, nd d7 of UUO. Po.5 vs. respective control; n ¼ 5. The mounts of mplifiction products for COX-1, COX-, nd EP mrnas were normlized to tht for 18S rrna, nd the vlue in control kidney is presented s 1. Control, kidneys from nonoperted mice. on dy 1 of UUO compred with tht in the control kidney nd remined to similr extent on dys nd 7 of UUO (Figure 1c), suggesting tht COX--derived prostnoids ply role in the pthogenesis of tuulointerstitil firosis fter UUO. Interestingly, significnt increse in the expression level of mrna for COX-1, constitutive isoform of COX, ws lso found in the UOK on dys nd 7 of UUO (Figure 1c). Next, we exmined the expression levels of mrnas for EPs in UOK efore nd fter UUO. Before UUO, mrnas for ll d1 d d7 Kidney Interntionl (1) 8,

3 originl rticle N Nkgw et l.: Prostglndin E in renl firosis EPs were detected. After UUO, the expression level of EP mrna incresed significntly nd reched level of up to sevenfold on dy 7 of UUO (Figure 1d), suggesting n EP - medited role of PGE in the development of tuulointerstitil firosis. Although the expression level of EP mrna incresed significntly to level of up to twofold on dy 7 of UUO, those of EP 1 nd EP 3 mrnas did not chnge or decresed significntly, respectively (Figure 1d). There were no significnt differences in the expression levels of EP mrna etween the upper nd lower prts of the kidney efore nd 7 dys fter UUO (dt not shown). On the sis of these results, we set out to clrify the EP - medited role of PGE in tuulointerstitil firosis using the UUO model in the following exmintions. c UOK EP / UOK d CLK UOK (+ ONO-819) Augmented tuulointerstitil firosis in EP mice nd EP -medited suppression of tuulointerstitil firosis y ONO-819 in mice To ssess the degree of firotic response to UUO, interstitil collgen deposition ws exmined histologiclly in Sirius red stined renl sections, nd hydroxyproline content ws mesured for quntifiction of firosis. In the kidneys of mice ( kidneys), tuulointerstitil firosis developed progressively fter UUO, nd renl hydroxyproline content incresed twofold over tht of control kidneys on dy 7 of UUO (Figure nd e). In the kidneys of EP mice (EP kidneys), however, tuulointerstitil firosis ws significntly ugmented compred with tht in kidneys on dy 7 of UUO (Figure c nd e), indicting tht endogenous PGE hs suppressive role in tuulointerstitil firosis vi EP. Furthermore, when EP ws stimulted y dministrtion of ONO-819, n EP -specific gonist, the degree of tuulointerstitil firosis in kidneys ws significntly reduced (Figure d nd e). In ddition, the effect of ONO-819 ws completely olished in EP kidneys (Figure e), indicting tht potent suppressive effect of ONO-819 on tuulointerstitil firosis ws medited y EP. There ws no pprent increse in interstitil collgen deposition in the contrlterl kidney (CLK) on dy 7 of UUO (Figure ). Augmented mcrophge infiltrtion nd myofirolst prolifertion in EP kidneys nd suppressive effects of ONO-819 on these ltertions In tuulointerstitil firosis, mcrophge infiltrtion is chrcteristic feture of locl inflmmtion, nd infiltrted mcrophges hve criticl role in the succeeding myofirolst prolifertion nd firosis. 7,8,1 Therefore, we next exmined mcrophge infiltrtion into the renl interstitil re in the UUO model (Figure 3). In kidneys, UUO induced progressive mcrophge infiltrtion, which ws noted exclusively in the perituulr re (Figure 3 nd e). This infiltrtion of mcrophges ws detected s erly s dy 1 of UUO (dt not shown). In EP kidneys, however, the degree of mcrophge infiltrtion ws significntly greter thn tht in kidneys on dys nd 7 of UUO (Figure 3c nd e), suggesting n EP -medited inhiitory effect of endogenous e Hydroxyproline (μg/mg kidney) ONO-819 C d7 d7 PGE on mcrophge recruitment. In ddition, ONO-819 significntly suppressed the UUO-induced mcrophge infiltrtion in kidneys, n effect sent in EP kidneys (Figure 3d nd e). In control kidneys nd CLKs, smll numer of mcrophges were detected in the interstitil re, ut there ws no significnt difference in the mcrophge numer etween nd EP kidneys (Figure 3 nd e). In ccordnce with mcrophge infiltrtion, UUO induced remrkle prolifertion of interstitil myofirolsts in kidneys fter UUO (Figure nd e). C + + Figure Renl interstitil firosis in wild-type () nd EP mice fter unilterl ureterl ostruction (UUO). ( d) Representtive photomicrogrphs of Sirius red stined tissue sections from, EP, nd ONO-819-treted kidneys on dy 7 (d7) of UUO. Interstitilly deposited collgen fiers nd the sement memrne of renl tuules were stined red. Originl mgnifiction. (e) The effect of ONO-819 on the renl hydroxyproline content fter UUO. Control (C), kidneys from nonoperted mice. Po.5; n ¼ Brs ¼ mm. CLK, contrlterl kidney;, not significnt; UOK, ureter-ostructed kidney. 16 Kidney Interntionl (1) 8,

4 N Nkgw et l.: Prostglndin E in renl firosis originl rticle UOK CLK UOK CLK c EP / UOK d UOK (+ ONO-819) c EP / UOK d UOK (+ ONO-819) e Cell counts/hpf (+ ONO-819) (+ ONO-819) In EP kidneys, however, myofirolst prolifertion ws significntly ugmented compred with tht in kidneys on dys nd 7 of UUO (Figure c nd e). As nticipted, ONO-819 significntly suppressed the UUOinduced myofirolst prolifertion in kidneys, n effect sent in EP kidneys (Figure d nd e). In control kidneys nd CLKs, myofirolsts could not e found in the interstitil spce (Figure nd e). These results indicte tht the PGE EP system negtively regultes mcrophge infiltrtion, therey succeeding myofirolst prolifertion fter UUO. The results therefore suggest the involvement of chemokine(s) for mcrophges s the trget molecule of the system. Control CLK d7 UOK d UOK d7 Figure 3 Interstitil infiltrtion of mcrophges in wild-type () nd EP kidneys fter unilterl ureterl ostruction (UUO). ( d) Representtive photomicrogrphs of tissue sections showing interstitil mcrophge infiltrtion in, EP, nd ONO-819-treted kidneys on dy 7 (d7) of UUO. F/8- positive mcrophges were stined rown. Originl mgnifiction. (e) Interstitil infiltrtion of mcrophges in the kidneys on d nd d7 of UUO. The extent of mcrophge infiltrtion ws expressed s interstitil mcrophge numer per high-power field ( ). Po.5 vs. mice; n ¼ 5 7. Brs ¼ mm. CLK, contrlterl kidney; HPF, high-power field;, not significnt; UOK, ureter-ostructed kidney. e Interstitil myofirolsts (%) (+ ONO-819) (+ ONO-819) Expression levels of MCP-1 nd RANTES mrnas re significntly higher in EP kidneys thn in kidneys fter UUO MCP-1 nd RANTES re thought to e the mjor chemokines responsile for mcrophge recruitment into the renl interstitium under inflmmtory conditions. 7,8 Therefore, we exmined the renl expression of MCP-1 nd RANTES mrnas fter UUO. In kidneys, expression levels of MCP-1 nd RANTES mrnas incresed remrkly fter UUO (Figure 5). In EP kidneys, however, the expression levels of these mrnas were significntly Control CLK d7 UOK d UOK d7 Figure Interstitil myofirolst prolifertion in wild-type () nd EP kidneys fter unilterl ureterl ostruction (UUO). ( d) Representtive photomicrogrphs of tissue sections showing interstitil myofirolst prolifertion in, EP, nd ONO-819-treted kidneys on dy 7 (d7) of UUO. Myofirolsts positive for -smooth muscle ctin (-SMA) were stined rown. Originl mgnifiction. (e) Interstitil myofirolst prolifertion in the kidney on d nd d7 of UUO. The re occupied y specificlly stined myofirolsts ws expressed s percentge of the totl re excluding the tuulr luminl re. Po.5 vs. mice; n ¼ 5 7. Brs ¼ mm. CLK, contrlterl kidney;, not significnt; UOK, ureterostructed kidney. Kidney Interntionl (1) 8,

5 originl rticle N Nkgw et l.: Prostglndin E in renl firosis 3 1 mrna/gapdh MCP-1 / EP (+ ONO-819) (+ ONO-819) mrna/gapdh TGF-β1 (+ ONO-819) / EP (+ ONO-819) Control CLK d7 UOK d UOK d7 1 Control CLK d7 UOK d UOK d7 mrna/gapdh 8 6 RANTES / EP (+ ONO-819) (+ ONO-819) mrna/gapdh CTGF (+ ONO-819) / EP (+ ONO-819) Control CLK d7 UOK d UOK d7 Figure 5 Expression of MCP-1 (monocyte chemottrctnt protein-1) nd RANTES (regulted on ctivtion, T cell expressed, nd secreted) mrnas in the kidney fter unilterl ureterl ostruction (UUO). Expression levels of () MCP-1 nd () RANTES mrnas in the kidney on dy (d) nd dy 7 (d7) of UUO. The mounts of mplifiction products for MCP-1 nd RANTES mrnas were normlized to tht for glycerldehyde-3- phosphte-dehydrogense (GAPDH) mrna. Po.5 vs. wild-type () mice; n ¼ 5 7. CLK, contrlterl kidney;, not significnt; UOK, ureter-ostructed kidney. Control CLK d7 UOK d UOK d7 Figure 6 Expression of trnsforming growth fctor-1 (TGF-1) nd connective tissue growth fctor (CTGF) mrnas in the kidney fter unilterl ureterl ostruction (UUO). Expression levels of () TGF-1 nd () CTGF mrnas in the kidney on dy (d) nd dy 7 (d7) of UUO. The mounts of mplifiction products for TGF-1 nd CTGF mrnas were normlized to tht for glycerldehyde-3-phosphte-dehydrogense (GAPDH) mrna. Po.5 vs. wild-type () mice; n ¼ 5 7. CLK, contrlterl kidney;, not significnt; UOK, ureter-ostructed kidney. higher thn those in kidneys on dys nd 7 of UUO (Figure 5), indicting n EP -medited suppressive ction of endogenous PGE on the UUO-induced chemokine induction. Accordingly, ONO-819 significntly suppressed the UUO-induced renl expression of MCP-1 nd RANTES mrnas in mice, n effect sent in EP mice (Figure 5). In control kidneys nd CLKs, there were no significnt differences in the expression levels of these chemokine mrnas etween nd EP mice. There were no significnt differences in the expression levels of RANTES mrna etween the upper nd lower prts of the kidney efore nd 7 dys fter UUO in oth nd EP mice (dt not shown). These results indicte tht the PGE EP system indeed suppresses chemokine induction nd limits the progression of tuulointerstitil firosis. Augmented expression of TGF-1 nd CTGF mrnas in EP kidneys fter UUO Recruited mcrophges, s well s tuulr epithelil cells, re the mjor source of TGF-1 nd CTGF, the representtive profirotic growth fctors responsile for firolst prolifertion nd for the differentition of firolsts into myofirolsts. 16, Becuse mcrophge recruitment ws significntly ugmented in EP kidneys, we next exmined whether renl production of these mcrophge-derived growth fctors increses in EP mice fter UUO. In kidneys, the expression levels of TGF-1 nd CTGF mrnas incresed remrkly fter UUO (Figure 6). In EP kidneys, however, the expression level of TGF-1 mrna ws significntly higher thn tht in kidneys on dy 7 of UUO (Figure 6). The expression level of CTGF mrna in EP kidneys ws lso significntly higher thn tht in 16 Kidney Interntionl (1) 8,

6 N Nkgw et l.: Prostglndin E in renl firosis originl rticle Shm UOK Figure 7 Locliztion of cyclooxygense- (COX-) nd monocyte chemottrctnt protein-1 (MCP-1) proteins in wild-type () kidneys efore nd fter unilterl ureterl ostruction (UUO). COX-- nd MCP-1-costined sections (upper pnels) nd corresponding hemtoxylin nd eosin stined sections (lower pnels) of the () control kidney nd the () ureter-ostructed kidney (UOK) on dy 7 of UUO. Immunorectivities for COX- were detected round the nuclei (red color) nd those for MCP-1 within the cytosol (green color). Brs ¼ 5 mm. kidneys on dys nd 7 of UUO (Figure 6), confirming n EP -medited suppressive ction of endogenous PGE on the UUO-induced induction of profirotic growth fctors. As nticipted, ONO-819 significntly suppressed the UUO-induced expression of TGF-1 nd CTGF mrnas in kidneys, n effect sent in EP kidneys (Figure 6). In control kidneys nd CLKs, there were no significnt differences in the expression levels of these growth fctors etween nd EP mice. These results suggest tht the PGE EP system negtively regultes the TGF-1/CTGF xis in the development of tuulointerstitil firosis y reducing mcrophge recruitment. Expression of COX- nd MCP-1 proteins is loclized to renl tuulr epithelil cells fter UUO We next exmined y immunohistochemicl nlysis where the upregultion of COX- nd MCP-1 occurs within kidneys fter UUO. We found intense COX- immunorectivities minly in tuulr epithelil cells on dy 7 of UUO (Figure 7). Intense immunorectivities for MCP-1 were lso found minly in tuulr epithelil cells on dy 7 of UUO (Figure 7). Importntly, COX- nd MCP-1 immunorectivities were coloclized in some tuulr epithelil cells (Figure 7). In control kidneys, we could detect only slight immunorectivities for COX- nd MCP-1 (Figure 7). These results indicte tht renl tuulr epithelil cells re the mjor cells expressing oth COX- nd MCP-1 fter UUO nd suggest tht COX- -derived PGE is lso produced in tuulr epithelil cells. Expression pttern of EP mrna in kidneys fter UUO Next, we exmined the expression pttern of EP mrna within the kidney fter UUO y in situ hyridiztion nlysis. In control kidneys, EP mrna expression ws rely detectle (Figure 8). However, we could detect slight signls for EP mrna in the glomeruli nd vsculr wlls of control kidneys in other in situ hyridiztion nlyses using rdioisotope (dt not shown). On dy 7 of UUO, signls for EP mrna incresed remrkly throughout kidneys (Figure 8). Notly, these signls were loclized minly to tuulr epithelil cells nd to lesser extent to interstitil cells nd glomeruli (Figure 8c). To further confirm tuulr locliztion of EP mrna, E-cdherin, representtive mrker for tuulr epithelium, ws immunostined in contiguous tissue section (Figure 8d). Signls for EP mrnas were detected minly in the structure tht coincides with tuulr epithelium expressing E-cdherin (Figure 8d). There were no signls for EP mrna in EP kidneys on dy 7 of UUO (dt not shown). These results indicte tht the renl tuulr epithelium is the mjor site expressing EP fter UUO, while recruited mcrophges likely express EP. Effects of ONO-AE1-39 on TGF-1-induced EMT in cultured renl tuulr epithelil cells In renl tuulr epithelil cells, TGF-1 is representtive inducer of EMT, which ws chrcterized y chnges in the expression of multitude of proteins, such s -smooth Kidney Interntionl (1) 8,

7 originl rticle N Nkgw et l.: Prostglndin E in renl firosis c d Shm UOK UOK UOK muscle ctin (-SMA), nd trnscription fctors including Twist, s well s morphologicl chnges. We first exmined the effect of TGF-1 on the expression level of EP mrna in cultured renl tuulr epithelil cells isolted from kidneys. TGF-1 significntly incresed the expression level of EP mrna (Figure 9), suggesting upregultion of EP in response to proinflmmtory stimulus. Next, to test whether EP ctivtion ffects EMT, we exmined the effect of n EP -selective gonist ONO-AE1-39 on the TGF-1- induced expression of -SMA nd Twist mrnas in cultured renl tuulr epithelil cells. In oth tuulr epithelil cells isolted from nd EP kidneys, TGF-1 significntly incresed the expression levels of -SMA nd Twist mrnas (Figure 9 nd c), indicting induction of EMT. In tuulr epithelil cells from kidneys, ONO-AE1-39 significntly suppressed the TGF-1-induced expressions of -SMA nd Twist mrnas. In contrst, ONO-AE1-39 did not hve these effects on tuulr epithelil cells from EP kidneys (Figure 9 nd c), suggesting suppressive role of the PGE EP system in EMT. Effects of ONO-AE1-39 on PDGF-induced prolifertion of firolsts nd TGF-1-induced CTGF production y firolsts Next, we exmined whether EP works lso in renl firolsts s well s in tuulr epithelil cells. In cultured renl firolsts isolted from kidneys, ONO-AE1-39 significntly suppressed PDGF-BB-induced prolifertion in concentrtion-dependent mnner (Figure 1). In cultured renl firolsts isolted from EP kidneys, however, the suppressive effect of ONO-AE1-39 disppered (Figure 1). In ddition, ONO-AE1-39 significntly inhiited TGF-1-induced CTGF production y firolsts isolted from kidneys ut not tht y firolsts isolted from EP kidneys (Figure 1). These results indicte tht the PGE EP system would ply suppressive role in the development of tuulointerstitil firosis fter UUO cting on firolsts in ddition to tuulr epithelil cells. Figure 8 In situ hyridiztion nlysis of EP mrna expression in kidneys efore nd fter unilterl ureterl ostruction (UUO). (, ) Hemtoxylin nd eosin stined (left pnels) nd the corresponding hyridized sections (right pnels) tht show hyridiztion signls (green) for EP mrna in the () wild-type () control kidney nd () ureter-ostructed kidney (UOK) on dy 7 of UUO. (c) Higher mgnifictions of oxed res in. Arrows indicte positive signls detected in tuulr epithelil cells, nd n rrowhed indictes the signl in glomerulus. (d) E-cdherin immunostined (left pnel) nd the corresponding hyridized (right pnel) sections of UOK on dy 7 of UUO. E-cdherin immunorectivities nd EP mrna hyridiztion signls pper green. ( d) Nuclei pper lue with,6-dimidino- -phenylindole (DAPI) stining. Brs ¼ 1 mm. An EP gonist protects the kidney from folic cid induced renl injury Finlly, we exmined whether n EP gonist could protect the kidney in nother model of kidney disese s well, folic cid induced renl injury. Blood ure nitrogen nd serum cretinine concentrtions incresed significntly in folic cid injected l/c mice (Figure 11d nd e), indicting induction of renl injury y folic cid. ONO-819, however, significntly suppressed the increses in lood ure nitrogen nd cretinine concentrtions (Figure 11d nd e), suggesting the protective effect of ONO-819 in folic cid induced renl injury. In ddition, sustntil renl interstitil firosis ws oserved fter folic cid injection y histologicl exmintion with Sirius red stining (Figure 11 nd ); this firosis ws pprently suppressed y ONO-819 (Figure 11c). Accordingly, renl hydroxyproline content incresed significntly on dy 7 of folic cid injection (Figure 11f). Notly, ONO Kidney Interntionl (1) 8,

8 N Nkgw et l.: Prostglndin E in renl firosis originl rticle mrna/18s mrna/18s TGF-β1 AE1-39 mrna/18s 1 TGF-β1 AE1-39 c TGF-β1 AE1-39 EP α-sma Twist significntly suppressed the increse in renl hydroxyproline content (Figure 11f), indicting tht ONO-819 hs n ntifirotic effect lso in this model. DISCUSSION Suppression of the development of tuulointerstitil firosis hs emerged s n importnt issue for the mngement of EP / EP / Figure 9 Trnsforming growth fctor-1 (TGF-1)-induced upregultion of EP mrna nd n inhiitory effect of ONO- AE1-39 on TGF-1-induced epithelil mesenchyml trnsition (EMT) in cultured renl tuulr epithelil cells. Expression of () EP,() -smooth muscle ctin (-SMA), nd (c) Twist mrnas in renl tuulr epithelil cells in response to TGF-1 (5 ng/ml) in the presence or sence of ONO-AE1-39 (1 mmol/l). The expression levels of EP, -SMA, nd Twist mrnas were normlized to tht for 18S rrna. Po.5 vs. TGF-1 lone; n ¼ 7., not significnt. [ 3 H]-thymidine incorportion (% of control) AE1-39 (M) C CTGF/β-ctin (% of TGF-β1 stimultion) CTGF β-actin 3 1 TGF-β1 AE EP / EP / chronic kidney diseses ecuse it determines prognosis of the diseses, nd there is little ntifirotic therpy for preventing the progression of chronic kidney diseses to end-stge renl filure. Therefore, extensive reserch hs een conducted to clrify the mechnism of tuulointerstitil firosis, nd numer of signling molecules hve een proposed to prticipte in the pthogenesis of this condition. These signling molecules, such s chemokines, inflmmtory cytokines, nd profirotic growth fctors, medite the sequentil events tking plce during the development of tuulointerstitil firosis. 6,7,3 Among these meditors, TGF-1 nd CTGF constitute n estlished signling xis in the process of tuulointerstitil firosis. 15, In ddition, C Figure 1 Effects of ONO-AE1-39 on trnsforming growth fctor-1 (TGF-1)-induced prolifertion nd TGF-1-induced connective tissue growth fctor (CTGF) production in cultured renl firolsts isolted from mice on dy 7 of unilterl ureterl ostruction (UUO). () Prolifertion of firolsts ws estimted y the nucler incorportion of [ 3 H]-thymidine. Po.5 vs. respective control; n ¼ 6. () Effect of ONO-AE1-39 on TGF-1-induced CTGF production y firolsts. The representtive lots from five independent experiments re shown in the upper pnels. The expression levels of CTGF protein were normlized to tht for -ctin. C, control (no ddition of ONO-AE1-39);, not significnt;, wild type. Po.5 vs. TGF1 lone; n ¼ 5. Kidney Interntionl (1) 8,

9 originl rticle N Nkgw et l.: Prostglndin E in renl firosis c Control FA injection FA injection + ONO-819 d 3 1 ONO Control d7 e ONO Cretinine (mg/dl) severl fctors, such s ngiotensin II nd rdykinin, hve een reported s upstrem key molecules controlling the events. 8,1 However, the role of PGE s such key molecule remins to e determined. In EP kidneys, tuulointerstitil firosis ws significntly ugmented compred with tht in kidneys fter UUO, indicting tht endogenous PGE plys suppressive role in the progression of tuulointerstitil firosis vi EP.In ddition, when EP ws phrmcologiclly ctivted y n EP -specific gonist, ONO-819, UUO-induced tuulointerstitil firosis ws significntly diminished in kidneys. Furthermore, this effect ws completely sent in EP kidneys, indicting tht the potent suppressive effect of ONO-819 on tuulointerstitil firosis is medited y EP. To our knowledge, this is the first report demonstrting tht endogenous PGE nd n EP gonist exert suppressive ction vi EP on the development of tuulointerstitil firosis in vivo. The renl expression level of COX- mrna incresed significntly fter UUO, suggesting tht COX--derived BUN (mg/dl) f Hyp (μg/mg) ONO-819 Control Control d7 + Figure 11 Effects of ONO-819 on renl function nd interstitil firosis in folic cid (FA)-induced renl injury. FA (5 mg/kg) ws injected intrperitonelly to induce renl injury with or without sucutneous injections of ONO-819. ( c) Representtive photomicrogrphs of Sirius red stined tissue sections showing FA-induced interstitil firosis on dy 7 of the experiment. (d) Blood ure nitrogen (BUN), (e) serum cretinine concentrtion, nd (f) renl hydroxyproline (Hyp) content on dy 7 (d7) of FA injection. Po.5 vs. FA noninjected controls; Po.5 vs. ONO-819-nontreted controls; n ¼ 6. Brs ¼ mm. d7 prostnoids including PGE prticipte in the pthogenesis of tuulointerstitil firosis. This result is in good greement with reported results tht stte tht production of PGE increses significntly in the kidney fter UUO. 17,18,,5 In ddition, immunohistochemicl nlysis showed tht min COX- immunorectivities were loclized to tuulr epithelil cells fter UUO, suggesting tht PGE produced y COX- in tuulr epithelil cells exhiits some effects on neighoring cells, including tuulr epithelil cells themselves nd interstitil cells. In ddition, we found tht the expression level of EP mrna increses remrkly in kidneys fter UUO. It hs een reported tht EP mrna loclizes minly in the glomeruli nd the vsculr wll in norml kidneys. 6 After UUO, however, the expression of EP mrna ws detected minly in tuulr epithelil cells nd interstitil cells. Tken together, these results indicte tht the existence of the PGE EP system inherent in tuulr epithelil cells ecme pprent fter UUO nd suggest tht the system plys role in the development of tuulointerstitil firosis y cting on oth tuulr epithelil cells nd interstitil cells. Mcrophge infiltrtion hd egun s erly s dy 1 of UUO efore interstitil myofirolst prolifertion nd firosis ecme pprent (dt not shown), s reported previously. 7 In EP kidneys, mcrophge infiltrtion ws significntly ugmented compred with tht in kidneys fter UUO. Infiltrted mcrophges re known to produce profirotic growth fctors, such s TGF-1 nd CTGF, which stimulte resident firolsts to differentite into myofirolsts tht produce extrcellulr mtrices nd induce EMT, which supplies rich renewle source of myofirolsts. 1,1,8 Therefore, ugmented mcrophge infiltrtion ws suggested to e primrily responsile for the phenotypic expression in EP mice. Ureterl ostruction exposes tuulr epithelil cells to nonimmunologicl stress nd induces interstitil mcrophge infiltrtion y stimulting the production of tuulr epithelium-derived chemokines, such s MCP-1 nd RANTES. 7,8 Accordingly, the expression levels of MCP-1 nd RANTES mrnas were significntly higher in EP kidneys thn in kidneys fter UUO. In ddition, ONO-819 hd potent inhiitory effect on the expression of these chemokines. Furthermore, we found tht MCP-1 expression fter UUO ws loclized minly in tuulr epithelil cells of kidneys s reported previously. 9 Importntly, COX- nd MCP-1 immunorectivities were coloclized in some tuulr epithelil cells fter UUO, wheres these immunorectivities were rely detectle in control kidneys, indicting tht renl tuulr epithelil cells re the mjor cells expressing oth COX- nd MCP-1 fter UUO nd suggesting tht COX--derived PGE is lso produced in tuulr epithelil cells. These results indicte tht PGE cts on tuulr epithelil cells vi EP, inhiits the chemokine expression, nd thus suppresses mcrophge infiltrtion. Although the role of EMT in the development of tuulointerstitil firosis in vivo is questionle, 1,3 we exmined the expression of EP mrna nd the effects of EP gonist to confirm the ction of PGE vi EP on the tuulr 166 Kidney Interntionl (1) 8,

10 N Nkgw et l.: Prostglndin E in renl firosis originl rticle epithelium using cultured renl tuulr epithelil cells. TGF-1 could potently upregulte the expression of EP mrna in tuulr epithelil cells, suggesting mechnism of EP upregultion during the inflmmtory process. Furthermore, ONO-AE1-39 significntly inhiited TGF-1-induced EMT in cultured renl tuulr epithelil cells in n EP -dependent mnner, which grees well with the report tht PGE hs potent inhiitory effect on EMT in cultured Mdin-Dry cnine kidney cells. 31 This result indictes tht PGE indeed cts on tuulr epithelil cells vi EP nd ffects their function. On the other hnd, severl reports hve presented EP -medited effects of PGE on mcrophge function. These effects include EP -medited inhiitory effects of PGE on lipopolyscchride-induced expression of chemokines 3,33 nd inflmmtory cytokines 3 in cultured mcrophges, suggesting tht COX--derived PGE cts lso on mcrophges nd ffects the course of UUO-induced tuulointerstitil firosis. However, the importnce of these effects of PGE on mcrophge function during the development of tuulointerstitil firosis in vivo remins to e determined. Interstitil myofirolst prolifertion ws significntly ugmented in EP kidneys compred with kidneys fter UUO. As mcrophge recruitment ws significntly ugmented in EP kidneys, production of mcrophgederived growth fctors tht induce myofirolst prolifertion ws expected to e ugmented in EP kidneys fter UUO. Indeed, we found tht the expression levels of TGF-1 nd CTGF mrnas were significntly higher in EP kidneys thn in kidneys fter UUO nd tht ONO-819 exhiited potent inhiitory effect on the expression of these growth fctors in kidneys. This result supports the ide tht the PGE EP system suppresses myofirolst prolifertion fter UUO y inhiiting mcrophge recruitment, thus decresing the levels of mcrophge-derived growth fctors. However, in vitro inhiitory effects of PGE on firolst prolifertion nd collgen synthesis y firolsts 39, hve een reported. In ddition, we showed tht ONO-AE1-39 significntly inhiited the functions of cultured renl firolsts, PDGF-induced prolifertion, nd TGF-1-induced CTGF production in n EP -dependent mnner. Therefore, these inhiitory effects of PGE on firolst functions would prtly contriute to the suppressive role of the PGE EP system in the development of tuulointerstitil firosis. The PGE EP system hs een suggested to hve other ctions including proinflmmtory 1 nd vsculr ctions. For exmple, the PGE EP signling positively regultes the development of T helper type 1 nd type 17 susets nd determines the extent of immune inflmmtion. In reltion to the proinflmmtory ctions of COX--derived prostnoids, COX- inhiitor etodolc hs een reported to suppress tuulointerstitil firosis induced y UUO. 3 COX- nd EP hve een reported to e coloclized in the vsculture of dult humn kidney. However, the contriution of these proinflmmtory nd vsculr ctions to the development of tuulointerstitil firosis remins to e determined. Interestingly, ONO-819 significntly suppressed the development of interstitil firosis in folic cid induced renl injury. Although the precise mechnism of this ction remins to e clrified, this result further supports the ide tht the PGE EP system plys n ntifirotic role in kidney diseses ccompnied y interstitil firosis. Angiotensin II hs emerged s potent firogenic fctor under vrious pthologicl conditions, such s crdic hypertrophy 5 nd pulmonry firosis. 6 Mice lcking the ngiotensin II type 1 receptor hve een reported to exhiit reduced tuulointerstitil firosis nd interstitil mcrophge infiltrtion fter UUO, 7 phenotype tht is in shrp contrst to tht of EP mice shown in this study. In ddition, ngiotensin II hs een reported to increse the expression of chemokines nd inflmmtory cytokines, leding to ctivtion of inflmmtory cells 7,8 nd upregultion of TGF-1 expression, 8,1 indicting tht the PGE EP system would ply n opposite role to tht of the renin ngiotensin system in the pthogenesis of tuulointerstitil firosis. In contrst, mice lcking the rdykinin B receptor (B mice) hve een reported to exhiit ugmented tuulointerstitil firosis fter UUO. 9 Although this phenotype is similr to tht of EP mice, B mice hd significntly smller numer of renl interstitil mcrophges thn did mice fter UUO, suggesting different signling mechnism leding to suppression of tuulointerstitil firosis etween PGE nd rdykinin. In fct, signling of rdykinin hs een suggested to involve the ctivtion of plsminogen ctivtors nd the mtrix metlloproteinse- cscde, leding to degrdtion of the extrcellulr mtrix. 9 These results indicte tht two well-known inflmmtory meditors, PGE nd rdykinin, ply suppressive role in the development of tuulointerstitil firosis. In conclusion, this study clerly showed tht the PGE EP system is inherent in the renl tuulr epithelium tht expresses oth COX- nd EP in response to UUO. Thus, the nti-inflmmtory ction of the PGE EP system suppressing the tuulointerstitil firosis development took plce on the tuulr epithelium s well, in ddition to mcrophges nd firolsts, indicting the novel role of the tuulr epithelium of utilizing the PGE EP system in the development of tuulointerstitil firosis. MATERIALS AND METHODS Mice The genertion nd mintennce of EP mice hve een reported previously. 5 Most EP mice die postntlly s result of ptent ductus rteriosus nd do not survive t ll in the C57BL/6 ckground. Therefore, F progenies of EP mice tht survived nd their littermtes were mintined independently in the mixed genetic ckground of 19/Ol nd C57BL/6. 51 We could not completely exclude the possiility tht some fixtion of prticulr genes in nd EP mice occurs ecuse of selective reeding. To minimize the fixtion of prticulr genes, we generted on occsion new group of nd EP mice y crossing hetero mice otined y mting of nd EP mice. However, we elieve it necessry to consider the difference in genetic Kidney Interntionl (1) 8,

11 originl rticle N Nkgw et l.: Prostglndin E in renl firosis ckgrounds, especilly when exmining in vivo phenotype. All experiments, which were pproved y the Ashikw Medicl University Committee on Animl Reserch, were performed using 8- to 1-week-old femle mice. A model of renl interstitil firosis To induce the development of renl interstitil firosis, UUO ws performed ccording to the reported method. 7,9 Mice were nesthetized with ketmine (1 mg/kg intrperitonelly) nd xylzine (5 mg/kg intrperitonelly) nd were plced in the dominl position under ody temperture control. The left ureter ws exposed through smll suprpelvic incision nd ligted t two sites within the middle portion with 3/ silk thred. The ureter ws then cut etween the two ligtions to prevent retrogrde infection. At dys, 1,, nd 7 of UUO, the mice were killed nd the kidneys were removed. The upper hlf of the kidney ws fixed in % phosphte-uffered prformldehyde, followed y prffin emedding for histologicl nd immunohistochemicl exmintions. The lower hlf ws snp-frozen in liquid nitrogen nd stored t 8 1C until use for extrction of RNA. In some experiments, the upper hlf ws lso used for extrction of RNA to compre the expression levels of EP nd RANTES mrnas etween the upper nd lower prts of the kidney. When exmining the effects of ONO-819, specific EP gonist for in vivo use, 5 it ws dministered sucutneously t dose of.1 mg/kg shortly fter the ureter ligtion nd twice dy during the dys following the experimentl periods. The dministrtion ended dy efore kidney removl. The dose used hs een shown to e effective in severl in vivo models. 53 ONO-819 hs K i vlue of.7 nmol/l for [ 3 H]-PGE inding to cloned mouse EP nd n EC 5 vlue of 1.6 nmol/l for intrcellulr camp production in Chinese hmster ovry cells expressing cloned mouse EP. 5 ONO- 819 ws donted y Ono Phrmceuticl (Osk, Jpn). We exmined tuulointerstitil firosis induced in this model until dy 7 of UUO, ecuse intense firosis occurred therefter, resulting in destruction of norml tissue rchitecture nd hindering ccurte evlution. Becuse we could use limited numers of EP mice for the experiments, nd we excluded the kidneys tht did not show pprent hydronephrotic ppernce, the numer of mice in ech experimentl group ws vrile s presented in figure legends. Quntifiction of renl firosis On dy 7 of UUO or folic cid injection, the mice were killed nd the kidneys were removed. After the medull including the pelvis ws scrped off with scissors, the remining cortex ws snp-frozen in liquid nitrogen nd stored t 8 1C until hydroxyproline ssy ws performed. To estimte the degree of renl firosis, the hydroxyproline content of the renl cortex ws mesured colorimetriclly ccording to the reported method. 55 Histologicl nd immunohistochemicl exmintions Tissue sections of mm thickness were prepred from prffinemedded kidneys. For evlution of tuulointerstitil firosis nd folic cid induced renl firosis, the sections were stined with Sirius red. 9 For immunohistochemicl exmintion of mcrophge infiltrtion nd myofirolst prolifertion, deprffinized sections were incuted with 3% hydrogen peroxide for 1 min to lock endogenous peroxidse ctivity. The sections were then incuted for 3 min t room temperture with their respective primry ntiodies: nti-f/8 ntiody for mcrophges (T-8; BMA Biochemicls AG, Augst, Switzerlnd) nd nti--sma ntiody for myofirolsts (N158; DAKO, Glostrup, Denmrk). For negtive controls, non-immune ser were used s primry ntiodies. Tissue sections were then incuted with their respective iotinylted secondry ntiodies: nti-rt IgG ntiody (5537; BD PhrMingen, Sn Diego, CA) for F/8 nd nti-mouse IgG ntiody (K677; DAKO) for -SMA. Stining of immunocomplexes ws completed y incuting tissue sections with streptvidin peroxidse followed y 3,3 -diminoenzidine s sustrte, nd the sections were counterstined with hemtoxylin. Under light microscope t mgnifiction, 1 fields within the corticl re of ech section were nlyzed using Adoe PhotoShop softwre (Sn Jose, CA) ccording to the reported method. 9 The re occupied y specificlly stined myofirolsts ws expressed s percentge of the totl re excluding the tuulr luminl re. The degree of mcrophge infiltrtion ws presented s the numer of interstitil mcrophges per field. Costining of MCP-1 nd COX- ws performed in GeneticL (Spporo, Jpn) using nti-mcp-1 (7; Acm, Cmridge, UK) nd nti-cox- (1616; Cymn Chemicl, Ann Aror, MI) s primry ntiodies nd the Cy5- nd Cy3-conjugted tyrmide signl mplifiction system, respectively (PerkinElmer Life nd Anlyticl Sciences, Boston, MA). Contiguous sections were stined with hemtoxylin nd eosin. Rel-time PCR nlyses Totl RNA from kidney nd cultured cells ws isolted using Isogen (Nippon Gene, Toym, Jpn) or n RNesy firous tissue mini kit (QIAGEN GmH, Hilden, Germny). First-strnd cdnas were synthesized using SuperScript VILO (Invitrogen, Crlsd, CA). Rel-time PCR ws performed using the TqMn primers nd proes (Applied Biosystems, Foster City, CA) for COX-1 (Mm 771), COX- (Mm 7837), EP 1 (Mm 397), EP (Mm 3651), EP 3 (Mm ), EP (Mm 3653), -SMA (Mm 751), Twist (Mm 36), nd 18S rrna (Mm 39899). The cycling conditions of TqMn primers were s follows: 1 min t 95 1C, followed y 5 cycles of 15 s t 95 1C nd of 6 s t 6 1C. For the nlyses of MCP-1, RANTES, TGF-1, CTGF, nd glycerldehyde- 3-phosphte-dehydrogense (GAPDH) expressions, rel-time PCR ws performed using n SYBR Green ssy (Roche Dignostics, Indinpolis, IN) s reported previously. 51 The primer sets used were s follows: 5 -ACTGAAGCCAGCTCTCTCTTCCTC-3 (forwrd) nd 5 -TTCCTTCTTGGGGTCAGCACAGAC-3 (reverse) for MCP-1, 5 -ATATGGCTCGGACACCACTC-3 (forwrd) nd 5 -GATGCCGATTTTCCCAGGAC-3 (reverse) for RANTES, 5 - CGGGGCGACCTGGGCACCATCCATGAC-3 (forwrd) nd 5 - CTGCTCCACCTTGGGCTTGCGACCCAC-3 (reverse) for TGF-1, 5 -GAGTGGGTGTGTGACGAGCCCAAGG-3 (forwrd) nd 5 -ATGTCTCCGTACATCTTCCTGTAG T-3 (reverse) for CTGF, nd 5 -CGGCAAGTTCAACGGCACAGTCA-3 (forwrd) nd 5 - GGTTTCTCCAGGCGGCATGTCA-3 (reverse) for GAPDH. The cycling conditions of SYBR Green primers were s follows: 1 min t 95 1C, followed y cycles of 15 s t 95 1C, 5 s t 6 1C (MCP-1, TGF-1, CTGF, nd GAPDH) or 55 1C (RANTES), nd 15 s t 7 1C. In situ hyridiztion In situ hyridiztion ws performed in GeneticL using Qunti- Gene ViewRNA for the FFPE ssy kit with Pnomics protocols (Affymetrix, Snt Clr, CA). Tissue sections (5 mm) were prepred from prffin-emedded kidneys, deprffinized, oiled in pretretment solution, nd digested with proteinse K. Sections were hyridized for 3 h t 1C with designed proe ginst the mouse 168 Kidney Interntionl (1) 8,

12 N Nkgw et l.: Prostglndin E in renl firosis originl rticle EP (Affymetrix). Hyridized proes were mplified using PreAmp nd Amp molecules. Therefter, multiple leled proe oligonucleotides conjugted to lkline phosphtse were dded, nd FstRed sustrte ws used to produce signls (red fluorophore). Nuclei were stined with,6-dimidino--phenylindole. Contiguous sections were lso stined with hemtoxylin nd eosin. Stining of E-cdherin, typicl mrker for tuulr epithelil cells, ws performed using n nti-e-cdherin ntiody (3195; Cell Signling Technology, Beverly, MA) nd the Cy5-conjugted tyrmide signl mplifiction system (PerkinElmer Life nd Anlyticl Sciences). A series of high-resolution monochromtic imges were cptured using n utomted microscope pltform (PM-TM; HistoRx, New Hven, CT). Imges were then depicted y pseudocolor merging with imge processing softwre ImgeJ (Ntionl Institutes of Helth, Bethesd, MD). EMT using primry culture of mouse renl tuulr epithelil cells Renl tuulr epithelil cells were isolted from nd EP kidneys using n ntiody ginst Tmm Horsfll glycoprotein (THP), surfce mrker of the thick scending lim nd erly distl convoluted tuule, nd the mgnetic cell sorting system (Miltenyi Biotec, Bergisch Gldch, Germny). Briefly, kidneys were cut into smll pieces in Hnk s lnced slt solution t 1C nd incuted in the sme uffer contining 1 mg/ml collgense (Worthington Biochemicl, Lkewood, NJ) t 37 1C for 3 min. After wshing nd centrifugtion, cells were incuted with nti-thp rit ntiody (M-631, Snt Cruz Biotechnology, Snt Cruz, CA) for 15 min. After ddition of nti-rit-igg microeds, nti-thp-ntiodyound cells were seprted y pssge through mgnet column (Miltenyi Biotec). The collected THP-positive cells were incuted on type I collgen coted pltes with Dulecco s modified Egle s medium contining 1% fetl clf serum. Suconfluent cells were serum-strved y replcing the medium with Dulecco s modified Egle s medium contining.5% fetl clf serum nd 1 mmol/l indomethcin. After h, the cells were incuted with 5 ng/ml recominnt TGF-1 (R&D Systems, Minnepolis, MN) with or without n EP -selective gonist ONO-AE1-39 (1 mmol/l) 56 for h. ONO-AE1-39 ws donted y Ono Phrmceuticl. EMT ws estimted y expression of -SMA nd Twist mrnas using reltime PCR. Primry culture of mouse renl firolsts Renl firolsts were isolted ccording to the reported method, 57 with minor modifictions. In short, kidneys on dy 7 of UUO were treted with solution contining 1 mg/ml collgense. The relesed cells were hrvested nd filtrted through nylon mesh ( mm), nd then the cells were cultured in Dulecco s modified Egle s medium contining 1% fetl clf serum, 1 U/ml penicillin, nd 1 mg/ml streptomycin t 37 1C in5%co. Exmintion of renl firolst prolifertion Prolifertion of renl firolsts ws estimted y [ 3 H]-thymidine incorportion. The cells were plted into -well culture pltes t 1 cells per well. After 8 h of culture, the culture medium ws chnged to fresh one contining 1 mmol/l indomethcin. After h of serum strvtion, the cells were stimulted with 1 ng/ml PDGF- BB (Peprotech, Rocky Hill, NJ). ONO-AE1-39 (1 1 nmol/l) ws dded 6 min efore the ddition of PDGF-BB. After 18 h of culture, [ 3 H]-thymidine ws dded to the concentrtions of 37 KBq/ml, nd the cells were cultured further for 6 h. The mount of [ 3 H]- thymidine incorported into the cells ws quntified using liquid scintilltion counter. Western lotting for CTGF Mouse renl firolsts were plted into 6 mm culture dishes t 1 5 cells per dish. For TGF-1 stimultion, cells t B9% confluence were serum-strved in Dulecco s modified Egle s medium contining 1 mmol/l indomethcin. After h of serum strvtion, cells were stimulted with 1 ng/ml of recominnt TGF1. ONO-AE1-39 (1 nmol/l) ws dded 6 min efore the ddition of TGF-1. The cells were lysed in uffer contining mmol/l Tris/HCl (ph 7.5), 15 mmol/l NCl, mmol/l EDTA, 1 mmol/l DTT, 1% Triton X-1, mmol/l N 3 VO, 1 mmol/l NF, nd 1 mmol/l sodium pyrophosphte supplemented with protese inhiitor cocktil mixture (Sigm, St Louis, MO). Protein contents were mesured with BCA Protein ssy kit (Thermo Scientific, Rockford, IL). Smples were seprted y sodium dodecyl sulfte-polycrylmide gel electrophoresis, nd then proteins were trnsferred onto n Immoilon-P memrne (Millipore, Bedford, MA). After locking procedure, the memrne ws incuted overnight with nti-ctgf (699; Acm) or nti--ctin (Clone AC-15; Sigm) ntiody t 1C, followed y incution with secondry ntiody coupled to horserdish peroxidse. After wshing, the mount of CTGF or -ctin protein ws detected y enhnced chemiluminescence (GE Helthcre, Little Chlfont, UK). Signls of these lots were visulized using n LAS-3 imge nlyzer (Fuji Photo Film, Tokyo, Jpn). Folic cid induced renl injury On dy of the experiments, folic cid ws dministered intrperitonelly t dose of 5 mg/kg to 8-week-old mle l/c mice. 58 ONO-819 ws dministered sucutneously t dose of.1 mg/kg twice dy from dys to 6 of the experiments. To ssess renl function, lood ure nitrogen nd serum cretinine concentrtions were mesured using ssy kits (DIUR-5 nd DICT-5; BioAssy Systems, Hywrd, CA) on dy 7 of the experiment. The kidneys were then removed, nd the renl hydroxyproline content ws determined s descried. Sttisticl nlyses All vlues re expressed s mens±s.d. The comprison of more thn two groups ws nlyzed with one-wy nlysis of vrince followed y Bonferroni s test. For comprison of two groups, Student s t-test or the Mnn Whitney test ws conducted fter crrying out the F-test for equl vrince. Po.5 ws considered sttisticlly significnt. DISCLOSURE All the uthors declred no competing interests. ACKNOWLEDGMENTS We thnk K Nky nd T Yokoym for help in reeding nd mintennce of mice. We lso thnk K Nknishi nd Y Tkshim for experimentl nd secretril ssistnce, respectively. This work ws supported y Grnts-in-Aid for Scientific Reserch from the Ministry of Eduction, Science, Sports nd Culture of Jpn nd y grnt from Core Reserch for Evolutionl Science nd Technology (CREST) of Jpn Science nd Technology Agency. This work ws lso supported y grnts from Ono Phrmceuticl, Tked Science Foundtion, Smoking Reserch Foundtion, nd Hokkido Hert Assocition. Kidney Interntionl (1) 8,