Kai Song 1,2,6, Fen Wang 1,6, Qian Li 1, Yong-Bing Shi 2, Hui-Fen Zheng 1, Hanjing Peng 3, Hua-Ying Shen 2, Chun-Feng Liu 1,4 and Li-Fang Hu 1,4,5

Transcription

1 & 213 Interntionl Soiety of Nephrology see ommentry on pge 1255 Hyrogen sulfie inhiits the renl firosis of ostrutive nephropthy PEN Ki Song 1,2,6, Fen Wng 1,6, Qin Li 1, Yong-Bing Shi 2, Hui-Fen Zheng 1, Hnjing Peng 3, Hu-Ying Shen 2, Chun-Feng Liu 1,4 n Li-Fng Hu 1,4,5 1 Institute of Neurosiene, Soohow niversity, Suzhou, Chin; 2 Deprtment of Nephrology, Seon Affilite Hospitl of Soohow niversity, Suzhou, Chin; 3 Deprtment of Chemistry n Center for Dignostis n Therpeutis, Georgi Stte niversity, Atlnt, Georgi, SA; 4 Deprtment of Neurology, Seon Affilite Hospitl of Soohow niversity, Suzhou, Chin n 5 Deprtment of Phrmology, Shool of Phrmeutil Siene, Soohow niversity, Suzhou, Chin Hyrogen sulfie hs reently een foun erese in hroni kiney isese. Here we etermine the effet n unerlying mehnisms of hyrogen sulfie on rt moel of unilterl ureterl ostrution. Compre with norml rts, ostrutive injury erese the plsm hyrogen sulfie level. Cystthionine--synthse, hyrogen sulfieprouing enzyme, ws rmtilly reue in the ureterl ostrute kiney, ut nother enzyme ystthionine--lyse ws inrese. A hyrogen sulfie onor (soium hyrogen sulfie) inhiite renl firosis y ttenuting the proution of ollgen, extrellulr mtrix, n the expression of -smooth musle tin. Menwhile, the infiltrtion of mrophges n the expression of inflmmtory ytokines inluing interleukin-1, tumor nerosis ftor-, n monoyte hemottrtnt protein-1 in the kiney were lso erese. In ulture kiney firolsts, hyrogen sulfie onor inhiite the ell prolifertion y reuing DNA synthesis n ownregulting the expressions of prolifertion-relte proteins inluing proliferting ell nuler ntigen n -My. Further, the hyrogen sulfie onor loke the ifferentition of quiesent renl firolsts to myofirolsts y inhiiting the trnsforming growth ftor-1-sm n mitogen-tivte protein kinse signling pthwys. Thus, low oses of hyrogen sulfie or its relesing ompouns my hve therpeuti potentils in treting hroni kiney isese. Kiney Interntionl (213) 85, ; oi:1.138/ki ; pulishe online 27 Novemer 213 KEYWRDS: ell signling; hroni inflmmtion; hroni renl isese Corresponene: Chun-Feng Liu, Deprtment of Neurology, Seon Affilite Hospitl of Soohow niversity, 155 Snxing Ro, Suzhou 2154, Chin. E-mil: liuf@su.eu.n or Li-Fng Hu, Institute of Neurosiene, Soohow niversity, 199 Ren-Ai Ro, Suzhou Inustril Prk, Suzhou , Chin. E-mil: hulifng@su.eu.n 6 Fen Wng n Ki Song re o-first uthors. Reeive 26 Jnury 213; revise 1 August 213; epte 29 August 213; pulishe online 27 Novemer 213 Tuulointerstitil firosis is the finl ommon pthwy to hroni kiney iseses (CKD). 1 Pthologilly, renl firosis mnifests s the formtion of myofilolsts n the eposition of extrellulr mtrix proteins in the renl interstitium. 2 The mehnisms of renl firosis hve not een fully eluite n effetive rugs re still sre. Renin ngiotensin losterone system is one of the min ulprits of renl firogenesis, n renin ngiotensin losterone system inhiitors remin the first-line rugs in fighting renl firosis. 3 However, the renin ngiotensin losterone system inhiitors my eteriorte renl funtion n use hyperpotssemi when serum retinine rises ove 3 mg/l. 4 New ntifiroti gents re therefore neee to expn therpeuti options n erese sie effets, whih is espeilly importnt for zotemi ptients. Hyrogen sulfie (H 2 S) represents the thir gsotrnsmitter long with nitri oxie n ron monoxie. 5 It is generte y ystthionine-g-lyse (CSE), ystthionine-synthse (CBS), n 3-merptopyruvte sulphurtrnsferse. CBS n CSE re enrihe in renl proximl tuules n proue H 2 S in kiney in omine wy. 6 H 2 S plys vrious physiologil n pthologil roles in the kiney. For instne, it exhiits iureti, ntriureti, n kliureti properties y inresing glomerulr filtrtion rte n funtions s n oxygen sensor in the renl meull. 6,7 Reently, it ws reporte tht plsm H 2 S level ws erese in 5/6 nephretomy rt n uremi ptients, 8,9 suggesting tht uremi toxin of CKD impirs the proution of enogenous H 2 S. Notly, the iologil funtions of H 2 S in CKD re not fully unerstoo. Heterozygous s / mie with unilterl nephretomy, n niml moel of CKD, evelope proteinuri n ollgen eposition, n inrese the expressions of mtrix metlloproteinse-2 n Emerging eviene lso emonstrtes tht H 2 S exhiits ntifiroti effets in the lung, hert, n liver Furthermore, H 2 S ers some similrities to the other two gseous moleules, nitri oxie n ron monoxie, oth of whih protet the kiney from renl firosis. 14,15 Tken together, we hypothesize tht H 2 S my ttenute renl firosis Kiney Interntionl (214) 85,

2 In this stuy, we exmine the effet of H 2 S on unilterl ureterl ostrutive () niml moel n efine its sfe n effetive osge rnge. Furthermore, we investigte the roles of H 2 S in renl firolst prolifertion n ifferentition. The potentil mehnisms were lso explore. RESLTS CBS expression n plsm H 2 S levels re erese in the ostrute kiney To investigte whether enogenous H 2 S ws involve in the pthogenesis of renl firosis, we exmine the expression n tivity of H 2 S-prouing enzymes CBS n CSE, s well s plsm H 2 S levels. The CBS expression ws nerly ompletely lte y ostrutive injury t y 14, wheres CSE ws inrese. In ontrst, the expressions of CBS n CSE in the ontrlterl kineys were not ffete (Figure 1 ). Moreover, H 2 S genertion in the ostrute kineys ws rmtilly reue ompre with shmoperte rts (Figure 1). Plsm H 2 S levels were reue y B3% in rts ompre with the shm ounterprts (27.5±4.3 mmol/l vs. 39.4±6.3 mmol/l, P ¼ 21, Figure 1e). Immunohistohemistry stining inite tht CBS ws preominntly expresse in proximl renl tuules. injury time-epenently reue CBS expression in the ostrute kineys without ffeting tht in the ontrlterl n shm-operte kineys (Figure 2 n ). In ontrst, CSE ws minly lote in renl glomeruli, interstitium, n interloulr rteries of norml rts. injury mrkely inrese the CSE expression in the interstitium of ostrute kineys. The CSE expression in the unostrute kineys remine unhnge (Figure 2 n ). NHS tretment ereses the renl size, inreses the ortil thikness, n meliortes renl funtion of rts To ssess the effet of exogenous H 2 S supplement on renl firosis, we trete rts with inrementl oses of soium hyrosulfie (NHS; 5.6, 56, n 56 mg/kg/y) n CSE inhiitor (DL-proprgylglyine (PAG), 25 mg/kg/y) vi intrperitonel injetion one ily for 3 ys efore n 2 weeks fter injury. The renin ngiotensin losterone system inhiitor enlpril (1 mg/kg/y) ws lso ministrte vi intrgstri s positive ontrol. Gross pperne (Figure 2) n group t (Figure 2 n ) showe tht enlpril n NHS (56 mg/kg/y) reue the size of the kiney n inrese the renl ortil thikness in rts. However, these effets were not oserve in the rts trete with NHS t higher ose of 56 mg/kg/y or PAG (Figure 3). Compre with rts, NHS (56 mg/kg/y) erese serum retinine levels, wheres PAG inrese serum ure nitrogen onentrtion. There were no ifferenes of serum eletrolytes mong eh group (Supplementry Dt n Supplementry Tle S1 online). NHS ttenutes renl tuulointerstitil injury n ollgen eposition in renl interstitium Hemtoxylin n eosin stining results inite tht rts exhiite ilte renl tuule, epithelil ells trophy, interstitil expnsion, n inrese infiltrtion of inflmmtory CBS CSE CBS fol inrese Contrlterl kiney strute kiney Shm Shm 63 k 36 k 44 k 36 k CSE fol inrese Shm Shm Contrlterl kiney e strute kiney Shm Shm Shm Shm Contrlterl kiney strute kiney Kiney H 2 S synthesis (μmol per g protein) strute kiney Figure 1 nilterl ureterl ostrutive () injury ownregultes ystthionine--synthse (CBS) expression n ereses hyrogen sulfie (H 2 S) level in plsm on y 14 fter opertion. () Representtive western lots of CBS n ystthionine-g-lyse (CSE) proteins in shm n group re shown. Reltive levels of () CBS n () CSE were nlyze y normlizing to glyerlehye-3- phosphte ehyrogense (). () Kiney H 2 S proution n (e) plsm H 2 S level in shm n group re shown. Dt re men±s.e.m., n ¼ 4 6, Po5, Po1, Po1 versus shm group. H 2 S level in plsm (μmol/l) Kiney Interntionl (214) 85,

3 Shm (3 ys) (7 ys) (14 ys) 6 CBS Contrlterl CBS-positive re (%) 4 2 strute Shm Shm Contrlterl kiney strute kiney CSE Contrlterl strute CSE-positive re (%) Shm Shm Contrlterl kiney strute kiney Figure 2 Expression n loliztion of ystthionine--synthse (CBS) n ystthionine--lyse (CSE) in rt renl ortex fter unilterl ureterl ostrutive () injury. (, ) CBS ws preominntly expresse in proximl renl tuules. CBS expression in the left (ostrute) kineys ws reue t 3 (3), 7 (7), n 14 ys (14) fter injury, wheres it remine unhnge in the right (ontrlterl) kineys. (, ) CSE ws minly lote in renl glomeruli, interstitium, n interloulr rteries n ws mrkely inrese in the left (ostrute) kineys t 3, 7, n 14 ys fter opertion. In ontrst, its expression in unostrute kineys ws not ltere. Sle r ¼ 2 mm. Dt re men±s.e.m., n ¼ 4, Po1 versus shm group, Po1 versus 3-y group, þþþ Po1 versus 7-y group. ells t 14 ys fter opertion. The tuulointerstitil injury of rts ws meliorte when trete with NHS (5.6 n 56 mg/kg/y) n enlpril. NHS t 56 mg/kg/y n PAG file to meliorte the tuulointerstitil injury ompre with group (Supplementry Dt n Supplementry Figure S1 online). Msson trihrome stining emonstrte tht injury resulte in signifint umultion of ollgen firils (lue re) ompre with the shm group. Enlpril n NHS (5.6 n 56 mg/kg/y) ttenute the firoti lesions with less ollgen eposite in the renl interstitium. In ontrst, NHS t 56 mg/kg/y n PAG inrese the ollgen proution ompre with tht of the group (Figure 4 n ). NHS inhiits the expression of SMA n fironetin in the ostrute kiney To evlute whether H 2 S reue the proution of myofirolsts n extrellulr mtrix, the renl expression of -smooth musle tin (-SMA) n fironetin were etete on y 14 fter opertion. Immunohistohemistry stining n semiquntittive nlysis showe tht enlpril n NHS (5.6 n 56 mg/kg/y) erese the expressions of -SMA n fironetin of the rts. In ontrst, NHS 56 mg/kg/y n PAG inrese the proution of these proteins, ut sttistil ifferene ws not otine in the PAG group (Figure 5 ). These oservtions were verifie y western lot nlysis, in whih NHS, espeilly t the ose of 56 mg/kg/y, mrkely reue the expressions of -SMA, fironetin, phospho-sm3, n trnsforming growth ftor-1 (TGF-1) (Figure 6 ). NHS inhiits inflmmtion in the kiney fter ostrutive injury The effet of H 2 S on the infiltrtion of inflmmtory ells in ostrute kineys ws lso etermine t 7 ys fter. CD68 ( mrophge mrker) stining in renl ortex revele n inrese mrophge infiltrtion in the renl interstitium of rts. NHS (5.6 n 56 mg/kg/y) n enlpril mrkely reue the numer of mrophge in the interstitium ginst the group. This effet ws most pronoune in the NHS (56 mg/kg/)-trete group. Compre with the group, NHS t 56 mg/kg/y inrese the numer of mrophge in renl ortex, wheres PAG tene ut file to signifintly inrese the CD68- positive ell numer (Figure 7 n ). To onfirm the nti-inflmmtory effet of NHS, the mrna levels of interleukin-1, tumor nerosis ftor-, n monoyte hemottrtnt protein-1 were etermine y quntittive PCR. All these inflmmtory moleules were 132 Kiney Interntionl (214) 85,

4 Shm NHS (5.6 μg/kg/y) Enlpril NHS (56 μg/kg/y) PAG NHS (56 μg/kg/y) Reltive length (left/right) 2. Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Cortex thikness (m) Shm Enlpril NHS (56 μg/kg/y) NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) PAG Figure 3 Soium hyrosulfie (NHS) tretment ereses the size n inreses the ortil thikness of unilterl ureterl ostrutive () kiney. () Generl pperne of four representtive left kineys of rts sujete to vrious tretments. rts reeive vehile (sline) tretment. NHS (5.6, 56, n 56 mg/kg/y) n DL-proprgylglyine (PAG; 25 mg/kg/y) were intrperitonelly given one ily 3 ys efore n ontinue for 2 weeks fter opertion, wheres enlpril (1 mg/kg/y) ws given vi intrgstri. () Reltive lengths of the left kiney lirte y the right ounterprt of the sme rt in ifferent groups. () Renl ortex thikness in the mi-portion of left kiney in oronl setion. Dt re men±s.e.m. (n ¼ 7), Po1 versus shm group; Po5, Po1 versus group. Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Are rtio of lue pixel re per high power (%) Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Figure 4 Soium hyrosulfie (NHS) tretment ttenutes the umultion of ollgen firils in renl interstitium in the ostrute kineys t 14 ys fter unilterl ureterl ostrutive () opertion. Representtive pitures of () Msson trihrome stining n () semiquntittive nlysis of the proportion with the lue olor re over the whole fiel re in ll groups re inite. PAG, DL-proprgylglyine. Sle r ¼ 2 mm. Men±s.e.m. (n ¼ 4), Po1 versus shm group; Po5, Po1 versus group. Kiney Interntionl (214) 85,

5 K Song et l.: Effet of H2S on Fironetin Shm Enlpril PAG Sh m N H En S l (5 p. N ril H 6 μ g/ S kg ( N 56 / H y μg S ) /k (5 g 6 / y μg ) /k g/ y) PA G NHS (56 μg/kg/y) NHS (56 μg/kg/y) NHS (5.6 μg/kg/y) Fironetin-positive re (%) α-sma PA G y) g/ μg 6 6 /k y) y) /k p S H N N H S (5.6 μg /k g/ l En (5 S H N g/ ril Sh μg PAG NHS (5.6 μg/kg/y) (5 NHS (56 μg/kg/y) Enlpril m NHS (56 μg/kg/y) Shm α-sma-positive re (%) Figure 5 Soium hyrosulfie (NHS) inhiits the expressions of fironetin n -smooth musle tin (-SMA) in the ostrute kineys t 14 ys fter unilterl ureterl ostrutive () opertion. Representtive immunohistohemistry imges for the expression of () fironetin n () -SMA, n semiquntittive nlysis of these two proteins re presente. PAG, DL-proprgylglyine. Sle r ¼ 2 mm. Dt re men±s.e.m. of 1 non-overlpping fiels from four nimls per group, Po1 versus shm group; Po5, Po1, Po1 versus group. inrese y injury. NHS (5.6 n 56 mg/kg/y) n enlpril suppresse the expression of these ytokines, wheres NHS t 56 mg/kg/y n PAG were unle to exhiit nti-inflmmtory effets (Figure 7). NHS suppresses the NRK-49F ell prolifertion inue y fetl ovine serum (FBS) We further performe n in vitro stuy to investigte the ntiprolifertion effet of H2S on renl firolst using NRK49F ells. Cells were expose to vrious onentrtions (1 5 mmol/l) of NHS for 3 min, followe y stimultion with 1% FBS for 24 h. Cells with serum-free meium were set s ontrols. MTT (3-(4,5-imethylthizol-2-yl)-2,5-iphenyltetrzolium romie) ssy showe the ell numer ws signifintly inrese fter inution with 1% FBS for 24 h (Po5, Figure 8). Preinution with NHS (1, 5, 1, n 5 mmol/l) reue the ell numer y 17.6%, 14.8%, 21.4%, n 16.2%, respetively. However, sttistil ifferene ws only rehe in 1 mmol/l NHS group n the ose-epenent effet ws not oserve. Furthermore, we ontinue to test whether H2S inhiite the DNA synthesis of renl firolst ells using romoeox1322 yuriine (Br) inorportion ssy. The DNA synthesis triggere y 1% FBS ws signifintly erese y NHS (1 mmol/l) efore tretment (Figure 8 n ). To elinete the mehnism eneth the ntiprolifertion effet of H2S, the expression of prolifertion-relte genes, inluing proliferting ell nuler ntigen (PCNA) n -My, ws lso ssesse. Preinution with 1 mmol/l NHS onsistently ttenute the expressions of PCNA n -My proteins inue y 1% FBS stimultion for 24 h (Figure 8 f). NHS loks the ifferentition of quiesent renl firolsts to myofirolsts Beuse TGF-1 is the most potent ytokine ontrolling the phenotype swith from renl firolsts to myofirolsts, we investigte the effet of H2S on renl firolst tivtion inue y TGF-1. Reverse trnsription PCR emonstrte tht NHS pretretment inhiite the mrna expressions of ollgen I, -SMA, n fironetin inue y TGF-1 (Figure 9 ). Western lot nlysis lso revele signifint elevtion of -SMA n fironetin expression in NRK-49F ells fter TGF-1 stimultion. These effets were essentilly olishe y NHS (Figure 9e n f). Kiney Interntionl (214) 85,

6 α-sma 43 K 25 K 36 K 36 K α-sma fol inrese fol inrese Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Fironetin 24 kd p-sm3 52 kd Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG 36 kd 36 kd Fironetin fol inrese Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG p-sm3 fol inrese Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Figure 6 Soium hyrosulfie (NHS) ownregultes the expression of firosis-ssoite n signling moleules in the ostrute kineys t 7 ys fter unilterl ureterl ostrutive () opertion. Whole kiney tissue lystes were immunolotte with ntioy ginst () -smooth musle tin (-SMA), () fironetin, () trnsforming growth ftor-1 (TGF-1), n () phospho-sm3 in eh group n quntittive nlyses of the reltive unne of these proteins mong vrious groups re presente., glyerlehye-3- phosphte ehyrogense; PAG, DL-proprgylglyine. Men±s.e.m., n ¼ 4, Po5, Po1 versus shm group; Po5, Po1, Po1 versus group. NHS olishes the phosphoryltion of the MAPKs n Sm3 TGF-1-Sm n mitogen-tivte protein kinse (MAPK) pthwys re two ritil signling mehnisms implite in renl firosis. We therefore exmine the effets of H 2 S on the TGF-1-inue phosphoryltion of Sm3, p38, -Jun N-terminl kinse (JNK), n extrellulr signl regulte kinse (ERK) with western lot nlysis. TGF-1 (2 ng/ml) triggere the phosphoryltion of these proteins with istint ptterns. The phosphoryltion of p38 peke t h fter TGF-1 stimultion, quiker thn tht of the other moleules. Speifilly, phosphoryltion of ERK n JNK peke t 2 h fter TGF-1 exposure wheres Sm3 t 1 h (t not shown). Pretretment with 1 mmol/l NHS olishe the inrese of Sm3 n MAPKs phosphoryltion inue y TGF-1 stimultion (Figure 1). DISCSSIN is well-estlishe niml moel feture y progressive renl firosis n tuulointerstitil injury. In this stuy, we emonstrte tht exogenous H 2 S suppresse ollgen n extrellulr mtrix eposition in the rts. We lso ientifie tht H 2 S loke renl firosis vi multiple mehnisms. It inhiite renl firolst prolifertion y reuing DNA synthesis n ownregulting the expressions of prolifertion-relte proteins. H 2 S lso loke ell ifferentition y suppressing the TGF-1-Sm n MAPK signl pthwys. Besies, H 2 S inhiite the inflmmtory proess inue y injury. The ssoition of H 2 S n renl firosis is not well efine. ur stuy emonstrte tht plsm H 2 S levels n enogenous H 2 S proution in the ostrute kineys were erese in rts. As moel is evoi of ny exogenous toxin, or uremi environment, 16 our fining implies tht renl firosis per se oul reue the H 2 S genertion. ur stuy further emonstrte tht CBS ws preominntly expresse in renl tuules wheres CSE ws minly lote in glomeruli n interstitil vessels, onsistent with reent report. 17 injury mrkely erese the expression of CBS ut inrese tht of CSE in the renl Kiney Interntionl (214) 85,

7 Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) Fol inrese to shm NHS (56 μg/kg/y) IL-1β Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Fol inrese to shm PAG MCP-1 Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Fol inrese to shm CD68-positive ell numer per fiel TNF-α Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Shm Enlpril NHS (5.6 μg/kg/y) NHS (56 μg/kg/y) NHS (56 μg/kg/y) PAG Figure 7 Soium hyrosulfie (NHS) inhiits the mrophge infiltrtion in the ostrute kineys t 7 ys fter unilterl ureterl ostrutive () opertion. () Representtive pitures of kiney setions stine with nti-cd68. A totl of 1 fiels from 4 nimls were inlue in nlyzing the numer of CD68-positive ells in the interstitium n () the group t re shown orresponingly. () Quntittive PCR results of the mrna levels of interleukin-1 (IL-1), tumor nerosis ftor- (TNF-), n monoyte hemottrtnt protein-1 (MCP-1) in the kineys mong ifferent tretment groups. PAG, DL-proprgylglyine. Sle r ¼ 1 mm. Dt re presente s men±s.e.m., Po1, Po1 versus shm group; Po5, Po1, Po1 versus group. interstitium. This fining is onsistent with previous stuies tht CBS is more preispose to erese in kiney isese ompre with CSE. For instne, CBS ws erese 6 weeks erlier thn CSE in rt remnnt kiney. 8 In renl ishemi reperfusion rts, CBS ws reue s erlier s 6 h fter reperfusion, 18 wheres CSE ws inrese y prolonge reperfusion for 24 h. 19 Hene, the inrese of CSE oserve in this stuy oul e expline s ompenstory mehnism to mintin the H 2 S level. As CSE is unnt in glomeruli, H 2 S my lso e enefiil for glomerulr iseses. A reent stuy y Lee et l. 2 onfirms this notion tht H 2 S is le to protet the renl glomerulr epithelil ells from high gluose inue injury, initing the reution of H 2 Sis involve in ieti nephropthy. As the enogenous H 2 S proution ws reue y injury, it is logil to hypothesize tht exogenous H 2 S supplementtion oul relieve renl firosis. We oserve tht the ntifiroti effet of NHS strte t 5.6 mg/kg/y, peke t 56 mg/kg/y, ut ws reverse t 56 mg/kg/y. ur results were in line with prior stuy in whih the most effetive NHS ose on ri ishemi reperfusion injury ws 56 mg/kg/y. 21 Moreover, low oses of NHS (78 n 392 mg/kg/y oy weight, twie ily) lso reue pulmonry firosis inue y leomyin. 11 Previous stuies emonstrte tht H 2 S level in verterte loo vrie from 1 3 mmol/l y the methylene lue ssy. 22,23 sing sensitive fluoresent proe, 24 we foun the plsm H 2 S level in norml rt ws B3 4 mmol/l, wheres its ounterprt in the rt ws 2 3 mmol/l. Nevertheless, the preise etermintion of H 2 S in iologil smples is still ontroversil. More sensitive tehniques suh s monoromoimne-se ssy n quntum ot/nnoprtile methos reently inite tht loo n tissue H 2 S onentrtions were s low s.15.9 mmol/l. 25,26 These finings my justify the pplition of H 2 S t reltively low oses in treting renl firosis. Although H 2 S is enefiil in most niml moels of renl iseses, enogenous H 2 S hs een reporte etrimentl in ute kiney injury use y nephrotoxi rugs. For instne, the CSE inhiitor PAG (5 mg/kg/y) reue the kiney mge of Wistr rts inue y ispltin n rimyin. 27,28 This is inonsistent with our t in whih PAG ws unle to relieve renl firosis. In ft, 5 mg/kg/y PAG tretment resulte in gret weight loss of norml Sprgue Dwley rts in our stuy (t not shown), n the ose h to e lowere to 25 mg/kg/y. Suh onfliting 1324 Kiney Interntionl (214) 85,

8 .4 Br DAPI Merge D vlue.3.2 CN.1 Br-positive ells (%) FBS NHS PCNA β-atin -My β-atin CN 1% FBS NHS 1 μmol/l 1% FBS NHS 1 μmol/l 1% FBS NHS 5 μmol/l 1% FBS NHS 1 μmol/l 1% FBS NHS 5 μmol/l 1% FBS CN FBS NHS NHS FBS 36 kd 43 kd 67 kd 43 kd e PCNA fol inrese FBS NHSFBS NHS CN 1% FBS NHS NHS 1% FBS f -My fol inrese 2. CN 1% FBS NHS NHS 1% FBS Figure 8 Soium hyrosulfie (NHS) suppresses the prolifertion of NRK-49F ells. Serum-strve ells were preinute with NHS t onentrtions s inite for 3 min, followe y stimultion with 1% fetl ovine serum (FBS) for 24 h. Serum-strve ells without FBS tretment serve s ontrols (CN). () MTT (3-(4,5-imethylthizol-2-yl)-2,5-iphenyltetrzolium romie) ssy shows the optil ensity (D) vlue of vrious tretment groups. (, ) Representtive mirogrphs (originl mgnifition 2, ) n () group t isply the numer of romoeoxyuriine (Br)-positive ells. Cells were stine with nti-br ntioy (re) n 4,6-imiino-2-phenylinole (DAPI; lue) ws use to visulize the nulei. N ¼ 6. ( f) Representtive pitures () n group t nlysis (e, f) for proliferting ell nuler ntigen (PCNA) n -My expressions in NRK-49F ells sujete to vrious tretments. Reltive unne ws etermine y normlizing to -tin. Dt re men±s.e.m. of three inepenent experiments. Po1, Po1 versus ontrol group; Po5, Po1, Po1 versus 1% FBS group. spets of H 2 S implite tht iverse mehnisms of H 2 S my exist in ifferent niml moels n speies. Despite this, ution must e tken in interpreting the effet of PAG euse of its nonseletive inhiitory tion of CSE. It hs een reporte tht D-mino i oxise n L-lnine trnsminse n lso e inhiite y PAG. 29,3 Possiilities nnot e rule out tht the protetive effet of PAG in renl isese my e meite y intivting other enzymes rther thn CSE. Firolst prolifertion n phenotypi trnsition to myofirolst re two mjor ellulr events of renl firosis. 31 FBS ontins numerous growth ftors tht promote the prolifertion n ifferentition in mny types of ells. The effet of H 2 S on ell prolifertion seems to e ell speifi. Some stuies reporte tht H 2 S promote the prolifertion in rt intestinl epithelil ells n humn olon ner ells, wheres others emonstrte tht it inhiite the prolifertion of lung firolsts n pnreti stellte ells. 32,33 ur t revele tht NHS t 1 mmol/l not only erese the ell numer, ut lso inhiite the DNA synthesis stimulte y FBS. These finings were further verifie y the ft tht NHS ownregulte the expressions of prolifertion-relte genes inluing PCNA n -My. Previous stuies lso inite tht H 2 S inue DNA mge in mny ell types. 34 The prolifertion n ell yle proteins suh s p53, p21, n ylin D1 were ownregulte y H 2 S. 35,36 Numerous ytokines n growth ftors suh s TGF-1, pltelet-erive growth ftor, tissue plsminogen tivtor, n plsminogen tivtor inhiitor-1 re involve in the phenotypi swith of firolst to myofirolst Mirorry nlysis in n intestinl ell line suggests tht TGF-1 n MAPK kinses re regulte y H 2 S. 41 ur results showe tht NHS t 1 mmol/l suppresse mrna Kiney Interntionl (214) 85,

9 Collgen I Fironetin NHS α-sma Fironetin mrna (fol inrese) CN TGF NHS TGF NHS 293 p 385 p 638 p 4 p Collgen-1 mrna (fol inrese) e NHS α-sma α-sma fol inrese CN TGF NHS TGF NHS CN TGF NHS NHS TGF 43 kd 36 kd α-sma mrna (fol inrese) f CN TGF NHS TGF NHS NHS Fironetin CN TGF NHS NHS TGF Figure 9 Soium hyrosulfie (NHS) pretretment loks the ifferentition of renl firolsts to myofirolsts. Serum-strve ells were inute with NHS (1 mmol/l) for 3 min, n then stimulte with trnsforming growth ftor-1 (TGF-1; 2 ng/ml) for 12 h (reverse trnsription PCR) or 24 h (western lot). ( ) Collgen I, fironetin, n -smooth musle tin (-SMA) mrna levels were etermine y () reverse trnsription PCR n ( ) semiquntifie y normlizing to glyerlehye-3-phosphte ehyrogense () y ensitometri nlysis. (e, f) Whole-ell lystes were immunolotte with ntioies ginst (e) -SMA n (f) fironetin, respetively. n -tin were loe s internl ontrols. Dt re men±s.e.m. of three inepenent experiments, Po5, Po1 versus ontrol group; Po5, Po1 versus TGF group. β-atin Fironetin fol inrese 24 kd 43 kd n protein expressions of -SMA n fironetin inue y TGF-1. In ition, H 2 S ttenute the phosphoryltion of Sm3, mjor pthwy in the signl trnsution of TGF-1. These t suggeste tht H 2 S inhiite the firolst ifferentition n extrellulr mtrix proution prtilly y loking the TGF-1-Sm signling. In ition to Sm-epenent pthwy, MAPK kinses lso engge in the effet of TGF-1. ERK, p38, n JNK hve een foun to e tivte in moel n regulte the prolifertion, migrtion, n ifferentition of firolst. 42 ur t inite tht H 2 S reverse the phosphoryltion inrese of p38, ERK, n JNK stimulte y TGF-1 in renl interstitil firolsts, implying tht the loke of MAPK kinses tivtion my e nother mehnism responsile for the ntifiroti effet of H 2 S. Inflmmtion plys n importnt role in the priming n progression of renl firosis. 43 ne of the pthologil fetures of the rt moel is the mrke infiltrtion of inflmmtory ells in the renl interstitium. Sustntil t support the nti-inflmmtion tion of H 2 S in multiple orgns n systems. 44 ur stuy ehoe with these stuies tht H 2 S exhiite n nti-inflmmtory property y reuing mrophge reruitment in renl interstitium. The inflmmtory ytokines suh s interleukin-1, tumor nerosis ftor-, n monoyte hemottrtnt protein-1 were lso ownregulte y NHS. MAPKs re involve in moulting inflmmtory proesses. For exmple, JNK hs een ientifie with the tivtion of mrophges n upregultion of proinflmmtory meitors, n JNK inhiition resolves the inflmmtion. 45 Thus, the ntiinflmmtory effet of H 2 S my e meite y inhiiting the MAPK signl pthwy. In summry, this stuy emonstrtes for the first time tht H 2 S exhiite ntifiroti effets on ostrute nephropthy n inhiite the prolifertion n ifferentition of renl firolsts oth in vitro n in vivo. The ntifiroti mehnisms of H 2 S my involve its nti-inflmmtion s well s its loke on TGF-1 n MAPK signling. This my provie therpeuti option for renl firosis se on H 2 S moultion. Low osge of H 2 S or its relesing ompouns my hve therpeuti potentils in treting CKD. MATERIALS AND METHDS Regents n ntioies Br, PAG, enlpril, n NHS, s well s ntioies ginst PCNA, -My, n Br were purhse from Sigm-Alrih (St Louis, M). Reominnt humn TGF-1 n Trizol regent were otine from Invitrogen (Crls, CA). Antioies ginst - SMA, fironetin, CBS, n ERK/phosphorylte-ERK were from Snt Cruz Biotehnology (Snt Cruz, CA). CSE ntioy ws purhse from Anov Chemil (Tiwn, Chin). ther ntioies unless speifie were from Cell Signling Tehnology (Dnvers, MA). Animl surgery n experimentl protools Mle Sprgue Dwley rts (18 2 g) were purhse from Shnghi Lortory Animl Commission (Shnghi, Chin) Kiney Interntionl (214) 85,

10 NHS NHS p-sm3 52 kd p-p38 43 kd 36 kd p38 4 kd Fol inrese (p-sm3/) Fol inrese (p-38/p38) CN TGF NHS NHS TGF CN TGF NHS NHS TGF NHS NHS p-jnk 46/54 kd p-erk 42/44 kd JNK 46/54 kd ERK 42/44 kd.25.8 Fol inrese (p-jnk/jnk) Fol inrese (p-erk/erk) CN TGF NHS NHS TGF CN TGF NHS NHS TGF Figure 1 Soium hyrosulfie (NHS) olishes the phosphoryltion inrese of mitogen-tivte protein kinse (MAPK) n Sm3. Serum-strve ells were inute with NHS (1 mmol/l) for 3 min, followe y trnsforming growth ftor-1 (TGF-1; 2 ng/ml) tretment for 1 h in the presene or sene of NHS. Cell lystes were sujete to western lotting with ntioies ginst () phospho- Sm3, () phospho-p38/p38, () phospho-jnk/jnk, n () phospho-erk/erk. Reltive unne ws quntifie y ensitometry n normlize to glyerlehye-3-phosphte ehyrogense (), p38, -Jun N-terminl kinse (JNK), n extrellulr signl regulte kinse (ERK), respetively. Dt re men±s.e.m., n ¼ 3, Po5, Po1, Po1 versus ontrol group; Po5, Po1 versus TGF group. opertion ws performe s esrie previously. 16 Rts were rnomly ivie into the following seven groups: shm,, with enlpril (1 mg/kg/y), with three oses of NHS (5.6, 56, n 56 mg/kg/y), n with PAG (25 mg/kg/y). NHS n PAG were given intrperitonelly one ily 3 ys efore surgery n ontinue for 1 to 2 weeks fter opertion. Enlpril ws given vi intrgstri. rts reeive vehile (sline) tretment. All rts were kille t y 7 or 14 ys fter surgery. The kineys were then hrveste n plsm ws ollete oringly. The experimentl protools were pprove y the Institutionl Animl Cre n se Committee of Soohow niversity. Histologil nlysis n immunohistohemil stining Kiney smples were fixe in 1% formlin, emee in prffin, n setione 4 mm thik. Setions were eprffine n stine with Msson trihrome regents n oserve uner light mirosope. Renl ollgen (lue re) ws semiquntittively mesure t 2 mgnifition in 1 rnomly selete fiels from 4 nimls using Imge Pro-Plus 6. softwre (Bethes, MD). For immunohistohemil stuies, renl setions were inute with ntioies ginst -SMA (1:5), fironetin (1:5), n CD68 (1:5) overnight t 4 1C, n susequently inute with orresponing seonry ntioies t room temperture for 1 h. Peroxise-Streptviin-iotin omplex (Vetor Lortories, Burlingme, CA) n iminoenziine (Sigm, St Louis, M) ws use to visulize the proteins. Ten rnomly selete fiels (2 mgnifitions for -SMA n fironetin, n 4 mgnifitions for CD68) were photogrphe n mesure y line oserver using Imge Pro-Plus 6. softwre. Plsm H 2 S n tissue H 2 S synthesis mesurement Plsm H 2 S level ws etete using Dnsyl zie (DNS-Az), novel fluoresent H 2 S proe (kinly gifte y Professor Binghe Wng, Georgi Stte niversity) s esrie previously. 24 Briefly, DNS-Az stok solution (2.2 mmol/l) ws e t 1 ml per well in 96-well plte, followe y mixing with 1 ml plsm. Fluoresene ws immeitely mesure y fluoresent miroplte reer (Ten M2, Groig, Austri) with n exittion t 36 nm n emission t 528 nm. Kiney Interntionl (214) 85,

11 Tissue H 2 S proution mesurement ws performe s previously esrie. 46 In rief, the left kiney homogentes were mixe with 2 mmol/l pyrioxl 5 -phosphte n 1 mmol/l L-ysteine n 25 ml 1% trihloroeti i to reh totl volume of 75 ml. After inution t 37 1C for 3 min, 25 ml 1% zin ette ws e to trp H 2 S. Then, 133 ml of 2 mmol/l N,Nimethyl-p-phenylenei-mine sulfte in 7.2 mol/l HCl n 133 ml of 3 mmol/l FeCl 3 in 1.2 mol/l HCl were e. The sorne of the solution ws etermine t 67 nm with spetrophotometer. The H 2 S onentrtion ws lulte ginst lirtion urve of NHS (.1 1 mmol/l). Cell ulture n tretments Norml rt kiney firolsts (NRK-49F) were purhse from Amerin Type Culture Colletion (Mnsss, VA) n mintine in Duleo s moifie Egle s meium (4.5 g/l gluose) supplemente with 1% fetl ovine serum (Gio, Grn Isln, NY). After preinution with NHS t vrious onentrtions (1 5 mmol/l) for 3 min, reominnt humn TGF-1 (2 ng/ml) ws e for vrious perios of time. In the ontrol group, ells were trete with vehile only. In prolifertion stuies, 1% FBS serve s stimultors n the ells were sujete to serum-free meium for 24 h efore experimenttion. Cell prolifertion ssy Cell prolifertion ws ssesse y MTT ssy n Br inorportion. For the MTT ssy, ells (21 3 /1 ml) were seee in 96-well plte until suonfluene n quiesene with serum-free meium for 24 h. Cells were then pretrete with NHS (.1 5 mmol/l) for 3 min, followe y inution with or without 1% FBS for 24 h. MTT ( mg/ml) ws e t 4 h efore tretment termintion. At the en of tretment, meium ws remove n imethylsulfoxie t 15 ml per well ws then e. Asorne t 49 nm ws mesure y miroplte reer (Ten M2). For the Br inorportion, ells were seee onto overslips. After eing pretrete with 1 mmol/l NHS for 3 min, ells were inute in meium with or without 1% FBS for 5 h, n pulste with Br (1 mmol/l) for nother 5 h. Next, ells were fixe in 4% prformehye for 2 min n DNA ws enture with 2 N HCl t 37 1C for 1 min. After loking, the inorporte Br ws etete with mouse nti-br ntioy (1:1, Sigm) t 4 1C overnight, followe y inution with Alex Fluor 555 Donkey nti-mouse IgG (1:5, Invitrogen) for 1 h. Susequently, overslips were mounte n oserve uner fluoresent mirosope (AXI SCPE A1; Zeiss, Jen, Germny). Reverse trnsription PCR n quntittive PCR Totl RNA ws extrte with Trizol regent n reverse trnsription PCR ws performe using n Avntge RT-for-PCR kit n PCR Mster Mix kit (Ferments, Vilnius, Lithuni) oring to the mnufturer s instrutions. The quntittive PCR retions were performe using rel-time SYBR tehnology n on ABI Prism 7 DNA Detetion System (Applie Biosystems, Foster, CA). For eh smple, glyerlehye-3-phosphte ehyrogense () n 18S RNA ws lso ssye s n internl ontrol n finl result were normlize to n expresse s rtios of the trget gene/internl ontrols. The primers use in the PCR retions re liste in Supplementry Tle S2 online. Western lot Given the heterogeneous expression of CBS n CSE in the kiney, the whole kiney ws hlve in the mile. ne hlf ws use for histologil stuies, n the other hlf ws homogenize for western lotting nlysis. Protein smples (2 8 mg) were seprte y 8 1% soium oeyl sulfte polyrylmie gel eletrophoresis gels n trnsferre onto polyvinyliene ifluorie memrne (Millipore, Befor, MA). After loking in 5% milk/tris-uffere sline n Tween-2 uffer, memrnes were iniviully inute with ntioies ginst proteins of interest t n pproprite ilution (-SMA (1:1), fironetin (1:5), CBS(1:5), CSE (1:25), ERK/phospho-ERK (1:5), JNK/phospho-JNK (1:5), P38/phospho-P38 (1:5), phospho-sm3 (1:5), PCNA (1:1), n -My (1:1)) t 4 1C overnight. Memrnes were then wshe n inute with pproprite horserish peroxise onjugte seonry ntioy for 1 h. Visuliztion ws rrie out using n vne hemiluminesene kit (GE Helthre, Bukinghmshire, K). The n ensity ws quntifie y ImgeJ softwre (Bethes, MD). or -tin ws use s n internl ontrol. Sttistil nlysis All t re presente s men±s.e.m. Sttistil signifine ws etermine using Stuent s t-test or one-wy nlysis of vrine followe y post ho nlysis (Tukey s test) where pplile. The signifine level ws set t Po5. DISCLSRE A ptent pplition on the potentil therpeuti option of H 2 S- relesing ompouns in renl firosis ( ) hs een file to Chin Intelletul Property ffie. All the uthors elre no ompeting interests. ACKNWLEDGMENTS We were grteful for the kin gift of Dnsyl zie y Professor Binghe Wng from Georgi Stte niversity. This work ws supporte y the reserh grnts from the Ntionl Nturl Siene Fountion of Chin (812495/212 to KS n to LF-H) n the strt-up funing for importe overses tlents of Soohow niversity (Q421521), n ws lso fune y projet fune y the Priority Aemi Progrm Development of Jingsu Higher Eution Institutions (PAPD). HP ws supporte y the Center for Dignostis n Therpeutis (CDT) progrm n Georgi Stte niversity Fellowship through the CDT. SPPLEMENTARY MATERIAL Tle S1. 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