Department of Pharmacology, Krishna teja pharmacy college, Tirupati, Andhra Pradesh, India 2

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1 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1, ORIGINAL ARTICLE Evlution of nti-firoti tivity of ethnoli extrt of Nelumo nuifer gertn. Seed ginst doxoruiin nd unilterl ureter ostrution-indued renl firosis R Rushendrn 1, Dr. V Jysnkr Reddy 2, T Bhrth Kumr 1, T Mmth, J Roj 1, T Roopvni 3 1 Deprtment of Phrmology, Krishn tej phrmy ollege, Tirupti, Andhr Prdesh, Indi 2 Professor, HOD, Deprtment of Phrmology, Krishn tej phrmy ollege, Tirupti, Andhr Prdesh, Indi 3 Deprtment of Phrmognosy, Sree Vidynikethn Phrmy College, Tirupti, Andhr Prdesh, Indi Rushendrn R, Jysnkr Reddy V, Bhrth Kumr T, Mmth T, Roj J, Roopvni T. Evlution of nti-firoti tivity of ethnoli extrt of Nelumo nuifer gertn. Seed ginst doxoruiin nd unilterl ureter ostrution-indued renl firosis. J Pre-Clin Clin Res. 217; 11(1): doi: /jpr/75319 Astrt Ojetive. The study were undertken to evlute nti-firoti tivity of ethnoli extrt of Nelumo nuifer seed (NNSEE) ginst doxoruiin nd Unilterl Ureter Ostrution-indued renl firosis. Mterils nd method. Animls were divided into five groups with six nimls in eh group, i.e. Norml, Positive ontrol, Stndrd (Pirfenidone 2mg/kg), Test-I (NNSEE 2mg/kg), nd Test-II (NNSEE 4mg/kg). Renl firosis ws developed y doxoruiin nd UUO-indued methods. After indution of renl firosis, profiroti responses in iohemil prmeters were oserved, e.g. BUN, serum retinine levels were elevted, while totl protein nd GFR levels deresed. Antioxidnt (SOD nd CAT) levels re lso ttenutes nd hylinized glomeruli ells, dmge to tuulr ells were noted in histopthology, whih re ll responsile for the development of renl firosis. Results. The result showed tht the nti-firoti tivity of NNSEE t dose of 2 nd 4mg/kg.wt ws omprle with the stndrd tretment 2mg/kg.wt of pirfenidone (nti-firoti drug). These dt supplemented with histopthologil studies of rt kidney setions. NNSEE hd reversed ll the mnifesttion of renl firosis. Conlusion. Results of the study indite tht the NNSEE hs potent nti-firoti tivity, s well s ntioxidnt property, in dose dependent mnner tht my e due to the presene of mjor phytohemil onstituents suh s lkloids, polyphenols. Key words renl firosis, nti-firosis, Nelumo nuifer, pirfenidone, kidney INTRODUCTION For humn eings, helth is most importnt from irth to end of life, ut from the pst to the present mn hs een suffering from diseses. Although glol moderniztion hs ought out revolutionry hnge in the field of mediine for dignosis, tretment nd understnding disese, ut the present dy sophistited life style, food hits, nture of work, et. re leding to numer of life-thretening diseses. Nowdys, renl firosis hs eome one of the most prolemti diseses hs een suffering throughout the world. Renl firosis ours when the norml tissue rhiteture is slowly tken ple y n extrellulr mtrix (ECM). Firosis of oth glomerulr nd tuulointerstitil omprtments orreltes with diminishing renl funtion in ll progressive forms of dieti nd non-dieti glomerulr disese whih leds to end-stge renl disese. Renl firosis is hrterized y the exessive deposition of onnetive tissue nd orgn inflmmtion. Literture reviews y the uthors of the presented study suggest tht the min reson for renl firosis hs reveled lose reltionship etween the expression of TGF-β nd ytokine. TGF-β is predominntly present in humn umilil vein endothelil ells. The min role of TGF-β is inresing the ECM protein synthesis, fironetin, Address for orrespondene: R Rushendrn, Deprtment of Phrmology in Krishn tej phrmy ollege E-mil: rushiyuv@gmil.om Reeived: 9 Jnury 217; epted: 19 My 217 nd lso promote the umultion of ECM. It ws reveled reently tht the TGF-β expression is upregulted in prolifertive nd firoti glomerulr lesions in vrious humns nd experimentl nephropthies, glomerulonephritis nd dieti nephropthy [1]. This ondition impts on millions of people worldwide nd n led to the deteriortion of the kidney, whih leds to deth. In ft, out 4% of ll deths due to kidney filure re linked to firosis. Kidney firosis typilly ours in every type of CKD, medil ondition hrterized y the grdul loss of kidney funtion over time [2]. Allopthi meditions re sfe nd effiious, ut prolonged usge my use disling side-effets. In order to remedy this kind of ondition, using the herl mediine is very effetive, rther thn the llopthi drugs. The World Helth Orgniztion (WHO) reports tht 8% of the world popultion presently use herl mediine for initil helth re. During the pst few dedes, the ffinity for herl drugs hs grown y the utiliztion of trditionl mediinl plnts to hel some ritil diseses. It is proving to e etter mediine with respet to syntheti drugs tht ssure numerous side-effets y long-term therpy. In reent yers, fous on plnt reserh hs inresed worldwide. A lrge ody of evidene hs een olleted tht shows the immense potentil of mediinl plnts used in vrious trditionl systems [3]. Although there re mny plnts like Apiumgrveolens, Boerhvidiffus, Phyllnthusniruri, Dtur metl whih hve mediinl properties, Hiisus sdriffet hs nephroprotetive property whih hs een proved sientifilly.

2 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of 67 Trditionlly using the Nelumo nuifer plnt, whih lso hs the nephroprotetive property, espeilly the seeds hving nti-firoti property hs not een sientifilly proved todte. Therefore, sed on the phytohemil onstituents present in Nelumo nuifer seed, n ttempt ws mde in the presented study to evlute the nti-firoti tivity ginst doxoruiin nd UUO-indued renl firosis, whih show more potent therpeuti effiy thn the present use of nti-firoti drugs. MATERIALS AND METHOD Plnt mterils nd uthentition. Dried seeds of Nelumo nuifer were olleted from n yurved store in Tirupti, Chittoor distrit, Andhr Prdesh, Indi, in Jnury 216. The plnt Nelumo nuifer ws identified nd uthentified y Dr. K. Mdhv Chetty, Assistnt Professor in the Deprtment of Botny t Sri Venkteswr University, Tirupti, Chittoor distrit, Indi. A vouher speimen ws sumitted in the Krishn Tej Phrmy College, Tirupti, Andhr Prdesh, Indi. The plnt mteril ws ir dried indoors t room temperture. Plnt extrtion. The olleted dried seeds of Nelumo nuifer were lened nd powdered in grinding mill. The seed powder ws shken in 44-mesh sieve for the orse fined powder. The fined powder ws stored in irtight ontiner t room temperture until preprtion of extrt. In the extrtion proess, two solvents were used petroleum ether nd ethnol. The fine-powdered drug ws sujeted to extrtion of petroleum ether solvent in 1:4 rtios (w/v), y using Soxhlet pprtus for 12 hours to remove ft mteril, nd the mr ws reused for extrtion with 7% ethnol t 4 C. The extrtion ws rried out until the extrt emes olourless. Extrt ws onentrted, dried nd pked in n ir-tight ontiner. The otined semi-solid extrt of Nelumo nuifer seed ws used for niml studies nd phytohemil sreening, stored t ool temperture of 2 4 C, throughout the durtion of the study [4]. Phytohemil estimtion. The preliminry phytohemil estimtion ws rried out y Pet. ether nd NNSEE for qulittive identifition of phytohemil onstituents, suh s lkloids, rohydrtes, gums, proteins nd mino ids, glyosides, steroids nd flvonoids y using stndrd proedures. ) Detetion of lkloids. Extrts were dissolved individully in dil. HCL nd filtered. Myer s test: Filtrte ws treted with Myer s regent (Potssium meruri Iodide) whih forms yellowoloured preipitte inditing the presene of lkloids. Wgner s test. Filtrte ws treted with Wgner s regent (Iodide in Potssium Iodide) whih forms rown or reddish-oloured preipitte inditing the presene of lkloids. ) Detetion of flvonoids Alkline regent test. Extrts were treted with few drops of sodium hydroxide solution whih turns yellow olour inditing the presene of flvonoids, nd y dding dilute id eomes olourless. Led ette test. Extrts were treted with few drops of led ette solution nd formed yellow olour preipitte inditing the presene of flvonoids. ) Detetion of rohydrtes. Extrts were dissolved individully in few ml of distilled wter nd filtered. Molish s test. Filtrtes were treted with two drops of loholi α-nphthol solution whih formed violet ring t the juntion, inditing the presene of rohydrtes. Benedit s test. Filtrtes were treted with Benedit s regent nd heted gently. An ornge red preipitte indited the presene of rohydrtes. d) Detetion of proteins nd mino ids. Xnthoprotei id. Extrts were treted with few drops of on. nitri id nd formed yellow olour inditing the presene of proteins. Ninhydrin test..25% w/v ninhydrin regent ws dded to the extrt nd oiled for few minutes, forming lue olour inditing the presene of mino id. e) Detetion of sponins. Forth test: Extrts were diluted with distilled wter (2ml) nd shken well in grduted ylinder for 15min. Formtion of 1m lyer of fom indited the presene of sponins. f) Detetion of tnnins. Geltin test. 1% geltin solution ontining sodium hloride ws dded to the extrt. A white preipitte formed inditing the presene of tnnins. g) Detetion of phenols. Ferri hloride test. Extrts were treted with 3 4 drops of ferri hloride solution whih formed luishlk olour inditing the presene of phenols. h) Detetion of glyosides. Modified Borntrger s test. Extrts were treted with ferri hloride solution nd immersed in oiling wter for pprox. 5 min. The mixture ws ooled nd extrted with n equl volumes of enzene. The enzene lyer ws seprted nd treted with mmoni solution whih formed rose-pink olour in the mmonil lyer, inditing the presene of glyosides. Legl s test. Extrts were treted with sodium nitropruside in pyridine nd sodium hydroxide nd hnged olour from pink to red, inditing the presene of glyosides. i) Detetion of Polysterols. Slkowski s test. Extrts were treted with hloroform nd filtered; the filtrte ws treted with few drops of on. sulfuri id, shken well, nd llowed it to stnd for few mins. The pperne of golden yellow olour indite the presene of polysterols. Lieermnn Buhrd s test. Extrts were treted with hloroform nd filtered; the filtrte ws treted with few drops of eti nhydride, oiled nd ooled. Con. sulfuri id ws dded whih formed rown ring t the juntion inditing the presene of polysterols (PrshntTiwriet l., 211).

3 68 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of Test nimls. Helthy Wistr lino rts (18 2g) were used in the study. Animls were proured from the niml house of our institute, limtized to lortory onditions, i.e. housed in polypropylene ges, 12:12hours light/drk yle, 24±2⁰C nd reltive humidity 5±5%. They were provide with stndrd feed pellet (Hindustn Lever Ltd., Bnglore) nd drinking wter d liitum throughout the study period (OECD Guidelines, 21). The nimls were fsted for 12 h prior to experimenttion. All the niml studies were performed ording to the rules nd regultions in the guidelines of the CPCSEA (Registered No. IAEC/1521/PO/Re/S/211/CPCSEA). Aute toxiity studies. An ute toxiity study ws rried out to demonstrte the pproprite sfe dose rnge tht ould e used for susequent experiments rther thn to provide omplete toxiity dt on the extrt. As per the OECD 423 Guidelines, Wistr lino rts (18 2g) were used for the toxiity studies. Helthy rts were rndomly ssigned to 4 groups with 3 rts in eh group. Rts were dministered with NNSEE of 5, 5, 3 nd 2,mg/kg,.wt. Signs or symptoms of ute toxiity nd mortlity rte were oserved ontinuously for the first 4 h, followed y 24 h, nd further oservtion prolonged for up to 14 dys. METHODOLOGY Doxoruiin induing method Body system. Antineoplsti nd Immunomodulting gents. Shedule: Chemotherpy type. Doxoruiin hydrohloride 1mg/5ml injetion, 5 ml vil. Doxoruiin hydrohloride 5mg/25ml injetion, 25 ml vil. Doxoruiin hydrohloride 1mg/5ml injetion, 5 ml vil. Animls re injeted intrvenously with single dose of doxoruiin 4 mg/kg. The rte of renl dmge nd development of firosis inreses with the inrese in doxoruiin dose. To exmine the extent of disese, this n e strtified on the sis of the proteinuri levels. The renl dmge strts fter 1 2 weeks nd progresses ontinuously for severl weeks. During this period of time, renl funtions drop nd ECM proteins grdully umulte in the glomeruli nd tuulointerstitium. At the end of the studied time, the nimls were srified nd the kidneys re exmined for tuulr injury, inflmmtion nd firosis using histopthologil nlysis. Biohemil prmeter suh s glomerulr filtrtion rte, serum retinine, lood ure nitrogen (BUN) nd totl protein levels were ssess. This model ws lso used to evlute with Pirfenidone (2mg/kg.wt, p.o) nd NNSEE. Unilterl ureter ostrution method. Animls were nesthetized with generl nesthesi with ketmine hloride (5mg/kg.wt i.p). The left kidney ws exposed vi flnk inision nd the ureter ligted with silk suture (i.e 4 ) for relile ligtion. Adominl musle nd skin were stithed seprtely, lyer-y-lyer. Renl firosis ws visile on dy 3 t the erliest, nd extensive firosis oured fter 2 3 weeks. This niml model ws from 3 dys to 21 dys, ut 14 dys is n optimum time for testing nti-firoti drugs. At the end of the studied time point, the nimls were srified nd the kidneys re exmined for tuulr injury, inflmmtion nd firosis, using histopthologil nlysis. Biohemil prmeters like glomerulr filtrtion rte, serum retinine, lood ure nitrogen (BUN) nd totl protein levels were ssess. This model hs een used to evlute with Pirfenidone (2mgkg,.wt) nd NNSEE[5]. Antioxidnt studies Ctlse (CAT). Ctlse tivity ws mesured y the method of Aei (1974). [6]. Clultion: Ctlse = log (A B) where: A Initil sorne B Finl sorne (fter 3 seonds) Units = μ moles of H 2 O 2 onsumed/min/mg Superoxide dismutse (SOD). Superoxide dismutse (SOD) tivity ws determined y the method developed y Kkkr et l, [7]. Clultion: SOD =.25 Y 1 Y 5 where: Y = Finl reding initil reding Histopthology. Kidney tissues were fixed in 1% formlin for t lest 24 hours, emedded in prffin nd ut into 3μm thik setions in rotry mirotome. The setion ws stined withhemtoxylin nd eosin, oserved under mirosope to detet histopthologil hnges in the kidney nd photogrphs were tken. Sttistil nlysis. Results were presented s men ± SEM (n=6). The dt ws sttistilly nlysed y One-wy nlysis of vrine (ANOVA), followed y Dunnett s multiple omprison tests (t test) nd P<.5, P<.1, P<.1 were onsidered s signifint, P>.5 ws onsidered s nonsignifint (ns) whih ws ompred with positive ontrol. All sttistil nlyses were performed with Grphpd Prism softwre pplition (7.1version). RESULTS Priliminry phytohemil test More phytohemil onstituents re present in NNSEE (lkloids, flvonoids, tnnins, sponins, rohydrtes, phenols, glyosides, proteins nd mino ids) thn in the Pet. ether extrt. Therefore, in the susequent studies NNSEE ws used euse it my show etter therpeuti effiy thn the petroleum ether extrt due to the presene of most of the iohemil onstituents. Aute toxiity studies The nimls were oserved individully fter dministrtion of NNSEE, t first 6 hours periodilly during the first 24 hours, nd therefter dily for totl 14 dys. There ws no

4 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of 69 hnge in the norml ehviourl pttern of rts; no signs nd symptoms of toxiity were oserved. NNSEE were sfe upto mximum dose of 2,mg/kg.wt. The iologil testing ws rried out t doses of 2 nd 4mg/kg.wt of NNSEE. The ute toxiity results of NNSEE re tulted elow. Biohemil prmeters S e r u m re tin in e le v e l ( m g /d L ) Grph 1. Estimtion of serum retinine level in doxoruiin-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Grph 1 shows the serum retinine level (mg/dl) mong different groups (n=6), ompred with positive ontrol (). Doxoruiin-indued in the rts resulted in signifnt inrese in serum retinine level, ompred with norml group. Elevted retinine level my led to one of the resonle profiroti responses. Rts were then treted with stndrd Pirfenidone (2mgkg,.wt) nd NNSEE (2mg/ kg nd 4mg/kg), whih ttenutes the profiroti responses. NNSEE nd pirfenidone, whih meliortes renl dysfuntion in rts ginst doxoruiin-indued renl firosis. Finlly, it ws oserved tht the test-2 of NNSEE showed etter results thn the test-1. B UN (m g /d L ) E ffet o f NNS E E o n seru m re tin in e le v e l g in st d o x o r ui in indued ren l fi r o sis D o x o r u i in ( 4 m g /k g ) P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) E ffet o f NNS EE o n B UN le v e l g in s t d o xor u i in indu e d ren l firosis Grph 2. Estimtion of BUN level in doxoruiin-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Grph 2 shows tht the BUN level (mg/dl) mong different groups (n=6) ompred with positive ontrol (). In this estimtion, when ompred with the norml group, the BUN level hd inresed in doxoruiin-indued rts. The norml BUN level my result in profiroti responses. Rts were then treted with stndrd (Pirfenidone 2mg/kg)nd NNSEE (2mg/kg nd 4mg/kg), whih ttenutes the elevted BUN levels. NNSEE nd pirfenidone meliortes renl dysfuntion in rts with doxoruiin-indued renl D o x o r u i in ( 4 m g /k g ) P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) firosis. Finlly, it ws oserved tht the test-2 dose of NNSEE showed etter results thn the test-1. Grph 3 shows the totl protein level (g/dl) mong different groups (n=6) ompred with positive ontrol (). Totl protein levels were deresed in doxoruiin-indued rts when ompred with norml group. Redued levels of totl protein ontent my led to profiroti responses. The rts were next treted with stndrd(pirfenidone 2mg/kg) nd NNSEE (2mg/kg nd 4mg/kg), whih meliortes the deresed levels of totl protein. NNSEE nd pirfenidone meliorte renl dysfuntion in rts with doxoruiinindued renl firosis. Finlly, it ws oserved tht the test- 2 dose of NNSEE showed etter results thn the test-1. G FR m L /m in /1.7 3 m 2 T otl protein level ( g /dl ) E ffet o f NNS E E o n T o t l p r o te in le v e l g in st d o x o r u i in indued ren l fi r o sis G ro up I (N o rm l) E ffe t o f N N S E E o n G F R le v e l g in s t d o x o r u i in G ro up II (Pos itive ontrol) indu ed ren l firosis G ro up III (S tndrd) G r o up I () G r o up II ( D o x o r ui in -4 m g /k g ) G r o u p I I I ( P ir fe n id o n e -2 m g /k g ) G r o up I V (N N S E E -2 m g /k g ) G r o u p V (N N S E E -4 m g /k g ) G ro up IV (T e s t-1 ) G ro up V (T e s t-2 ) Grph 4. Estimtion of GFR level in doxoruiin-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Grph 4 shows the GFR level (ml/min/1.73m 2 ) mong different groups (n=6) ompred with positive ontrol (). The GFR level signifntly deresed when treted with doxoruiin. Redued GFR level shows renl dysfuntion nd my led to profiroti responses. Rts were then treted with stndrd (Pirfenidone 2mg/kg) ndnnsee (2mg/kg nd 4mg/kg) whih reversed the effet of doxoruiin. NNSEE nd pirfenidone meliorte renl firosis in doxoruiinindued rts, inditing nti-firoti tivity. Finlly, it ws oserved tht the test-2 dose of NNSEE showed etter results thn the test-1. Grph 5 shows tht the serum retinine level (mg/dl) mong different groups (n=6) ompred with positive ontrol (). Serum retinine level ws signifntly rised due to ligtion of the ureter. An elevted retinine level my led to profiroti responses. The rts were then treted with stndrd (Pirfenidone 2mg/kg) nd NNSEE (2mg/kg D oxo r u i in ( 4 m g /k g ) P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) Grph 3. Estimtion of totl protein levels in doxoruiin-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test).

5 7 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of S e r u m re tin in e le v e l ( m g /d L ) E ffet o f NNS EE o n s eru m re tin in e le v e l g in s t U U O indued ren l fi r o sis UUO P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) Grph 5. Estimtion of serum retinine level inuuo-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). nd 4mg/kg) whih ttenutes the elevted levels inditig nti-firosis tivity. NNSEE nd pirfenidone meliorte renl dysfuntion in rts ginst UUO-indued renl firosis. Finlly, it ws notied tht the test-2 dose of NNSEE showed etter results thn the test-1. T otl protein level (g /dl ) E ffet o f NNS E E o n T o t l p r o te in le v e l g in s t UUO indu e d ren l fi r o sis UUO P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) Grph 7. Estimtion of Totl protein level in UUO indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One wy ANOVA followed y Dunnett s multiple omprisons test). E ffe t o f N N S EE o n G F R le v e l g in s t U U O indued renl firosis G r o up I () G r o u p I I (UUO ) G r o u p I I I ( P ir fe n id o n e -2 m g /k g ) G r o up I V (N N S E E -2 m g /k g ) 1 5 G r o u p V (N N S E E -4 m g /k g ) E ffe t o f NNS EE o n B U N le v e l g in s t U U O indued ren l fi r o sis U U O P ir fe n id o n e (2 m g /k g ) G FR m L /m in /1.7 3 m B UN (m g /d L ) NNSEE (2m g /kg) NNSEE (4m g /kg) Norml P os itive Control S tndrd Tes t-1 Tes t-2 G ro up I (N o rm l) G ro up II (P o s itiv e o ntro l) G ro up III (S t nd rd) G ro up IV (Te st-1 ) G ro up V (Te st-2 ) Grph 8. Estimtion of GFR level in UUO indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Grph 6. Estimtion of BUN level in UUO-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Grph 6 shows the BUN level (mg/dl) mong different groups (n=6) ompred with positive ontrol (). The BUN level ws signifintly rised fter ligtion of the right ureter. Elevted BUN level redues renl funtion whih leds to profiroti responses. The rts were next treted with stndrd (Pirfenidone 2mg/kg) ndnnsee (2mg/kg nd 4mg/kg) whih redues the elevted levels of BUN. NNSEE nd pirfenidone meliortes renl dysfuntion in rts with UUO indued renl firosis. Finlly, it ws oserved tht the test-2 dose of NNSEE showed etter results thn the test-1. Grph 7 shows the totl protein level (g/dl) mong different groups (n=6) ompred with positive ontrol () in UUO-indued rts when ompred with the norml group. Anorml levels of totl protein ontent my led to profiroti response. Rts were then treted with stndrd (Pirfenidone 2mg/kg) nd NNSEE (2mg/kg nd 4mg/kg) whih meliortes the deresed levels, inditing nti-firoti tivity. NNSEE nd pirfenidone meliortes renl dysfuntion in rts with UUO-indued renl firosis. Finlly, it ws noted tht the test-2 dose of NNSEE showed etter results thn the test-1. Grph 8 shows the GFR level (ml/min/1.73m 2 ) mong different groups (n=6) ompred with positive ontrol (). When ompred with the norml group, GFR level signifntly deresed in UUO-indued rts. An norml GFR level in urine indited renl dysfuntion whih my led to firosis. Rts were then treted with stndrd (Pirfenidone 2mg/kg) nd NNSEE (2mg/kg nd 4 mg/kg) whih meliortes the GFR levels. NNSEE nd pirfenidone(stndrd drug) meliortes renl firosis in rts, whih indites ntifiroti tivity. Finlly, it ws noted tht the test-2 dose of NNSEE showed etter results thn the test-1. S O D (U n its /m g p r o te in ) E ffe t o f N N S E E o n S O D le v e l g in s t d o x o r u i in indued renl firosis D o x o r u i in ( 4 m g /k g ) P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) Grph 9. Estimtion of SOD in doxoruiin-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Grph 9 shows the SOD (units/mg protein) level mong different groups (n=6) ompred with positive ontrol groups (). In the present study, the ntioxidnt itvity

6 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of 71 of Superoxide dismutse (SOD) enzyme (svenger of free rdils) in doxoruiin-indued rts ws investited. Free rdils re responsile for the profiroti responses. In the positive ontrol group, the SOD enzymes deresed, i.e superoxides were elevted, ompred to the norml group followed y the remining groups. Treting the rts with stndrd (Pirfenidone 2mg/kg) nd NNSEE extrt (2 nd 4mg/kg.wt) inreses the SOD enzyme whih ttenutes free rdil tivity. NNSEE nd pirfenidone meliortes renl firosis in doxoruiin-indued rts, inditing nti-firoti tivity dose dependently. Finlly, it ws oserved tht test-2 of NNSEE (4mg/kg.wt) showed etter therpeuti tion thn the test-1 (2mg/kg.wt). C A T (Units /m g protein ) E ffet o f N N S E E o n CAT le v e l g in st UUO indu ed ren l firosis Grph 1 shows the CAT (units/mg protein) level mong different groups (n=6) ompred with the positive ontrol group (). The presented study investigted the ntioxidnt itvity of tlse (CAT) enzyme (svenger of free rdils) in UUO-indued rts. Free rdils re responsile for the profiroti responses. In the positive ontrol group, the tlse enzyme deresed ompred to the norml group. Treting the rts with stndrd (Pirfenidone 2mg/kg) nd NNSEE extrt (2 nd 4mg/kg.wt) inresed the tlse enzyme whih ttenutes free rdil tivity. NNSEE nd pirfenidonemeliortes renl firosis in rts ginst UUO-indued rts indited nti-firoti tivity dose dependently. Ultimtely, notied tht test-2 of NNSEE (4mg/kg.wt) hs shown etter therpeuti tion thn the test-1 (2mg/kg.wt). HISTOPATHOLOGICAL STUDIES UUO P ir f e n id o n e (2 m g /k g ) N N S EE (2 m g /k g ) N N S EE (4 m g /k g ) Grph 1. Estimtion of CAT in UUO-indued renl firosis. Dt represent the men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Histopthology of UUO indued renl firosis Group I (Norml) Norml owmn s psule. No degenertive hnges. Norml yto-rhiteture kidney tissue. Group II (UUO) Degenertive hnges tkes ple in the kidney tissue nd shows nerosis. Vuoliztion ours. Hylinized glomeruli ells re oeserved. Inflmmtory ells re noted. Group III (Pirfenidone 2mg/kg.wt) Regenertive hnges tke ple in the kidney tissue nd shows similr to norml yto-rhiteture. Group IV (NNSEE low dose 2mg/kg.wt) Regenertive hnges took ple. Hylinized glomeruli ells were oserved.. NNSEE ttenuted pproximtely 75 8% of the hylinized glomeruli in kidney. Group V(NNSEE high dose 4mg/kg.wt) Regenertive hnges took ple in the kidney tissue nd showed similr to stndrd yto-rhiteture. Hylinized glomeruli were oserved lthough less ompred to the test-1 group. NNSEE ttenuted pproximtely 95% of hylinized glomeruli in kidney. NNSEE showed etter results thn the Test-1 group. UUO-indued kidney injury n led to progressive renl firosis. In the presented study, hylinized glomeruli ells or sr formtion ouring in the kidney ws ssessed y Hemtoxyline nd eosin stining. The result of UUOindued model is the prodution of mssive sr formtion of glomeruli ells oserved in the positive ontrol group. These firoti hnges in UUO injured kidney ws remrkly rogted y NNSEE nd stndrd drug, inditing ntifiroti tivity. Colletively, these dt suggest tht the NNSEE tretment improves the long-term outome of injured kidney y meliorting renl firosis. Histopthology of doxoruiin-indued renl firosis Group I (Norml) Norml Bowmn s psule. No degenertive hnges. Norml yto-rhiteture kidney tissue. Group II (UUO) Degenertive hnges took ple in the kidney tissue nd showed nerosis. Vuoliztion ourred. Tuulr nerosis or dmge oeserved. Inflmmtory ells noted. Group III (Pirfenidone 2mg/kg.wt) Regenertive hnges took ple in the kidney tissue nd showed similr to norml yto-rhiteture. Group IV (NNSEE low dose 2mg/kg.wt) Regenertive hnges took ple. Tuulr dmged ells oserved. NSEE ttenuted pproximtely 7 8% of tuulr nerosis in kidney. Group V(NNSEE high dose 4mg/kg.wt) Regenertive hnges took ple in the kidney tissue nd showed similr to stndrd yto-rhiteture. Tuulr nerosis were oserved lthough less ompred to the test-1 group. NNSEE ttenuted pproximtely 9% of tuulr nerosis in kidney. NNSEE showed etter results thn the test-1 group.

7 72 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of Doxoruiin-indued kidney injury n led to progressive renl firosis. The presented study, Hemtoxyline nd Eosin stining ws used to ssess tuulr dmge or tuulr neorosis ourring in the kidney. The doxoruiin-indued model produed mssive tuulr dmge in the positive ontrol group. These firoti hnges in doxoruiin-indued kidney ws remrkly rogted y NNSEE nd the stndrd drug, inditing nti-firoti tivity. Colletively, these dt suggest tht the NNSEE tretment improves the long-term outome of injured kidney y meliorting renl firosis. DISCUSSION In the presented study it ws oserved tht the tretment y NNSEE exerted enefiil effets on renl firosis indued with Doxoruiin nd Unilterl Ureter Ostrution (UUO) models. The results showed tht the umultion of firoytes, hyliniztion of glomeruli in the kidney ws evidently suppressed y NNSEE tretment. The possile underlying mehnism might e its inhiition of renl expression of TGF-β 1 nd MCP-1 hemottrtnts ontriuting to the firoytes reruitment into the injured kidney [8]. Renl firosis underlies ll forms of end-stge renl disese. It is the inevitle onsequene of n exessive umultion of extrellulr mtrix tht ours in virtully every type of hroni kidney disese [9]. The inidene of hroni kidney disese leding to end-stge renl disese (ESRD) ontinues to inrese worldwide. Almost ll forms of ESRD re preeded y the progressive pperne of renl firosis. Currently, tretment of renl firosis is severely limited nd often ineffetive [1, 11]. During the pst dede, mny drugs hve een introdued onto the mrket for the mngement of renl firosis; their effetiveness, however, ws unequivol mong the entire rnge of the popultion suffering from this renl disorder. Although most of these meditions re sfe nd effiious, in some ptients prolonged usge my hve questionle effiy nd use disling side-effets. This ft initites the development of nti-firosis drugs derived from herl soures nd whih hve etter rte of suess, ility to hieve full remission, with miniml inidene of side-effets. Alterntive tretments using herl mediines re therefore of gret interest. The use of herl mediines is now widespred for the tretment of vrious disese nd disorders. The presented study foused on most ommon nd hroni disese renl firosis, nd its tretment using herl extrts from trditionlly used herl plnt Nelumo nuifer. A study ws therefore designed to evlute the nti-firosis tivity in kidney y using NNSEE. Nelumo nuifer is hereous, quti plnt of the Nelumonee fmily, ntive to Tropil Asi nd Queenslnd, Austrli. It hs een used in folk mediine nd hs een proved to hve nti-oesity, nti-dieti, nti-fertility, heptoprotetive, pulmonry protetive, nti-prkinsonism, nti-teril, nti-oxidnt, nti-pltelet, nti-wrinkle, ytoprotetive, nlgesi, nti-prolifertive nd nti-inflmmtory properties [12]. It is lso used in the mngement of firosis ut its phrmologil mehnism on renl firosis remins unknown. Through ongoing investigtion in this re, the uthors of the presented study found tht the Nelumo nuifer seed hs nti-firoti tivity in kidney, lthough this is not yet een proved sientifilly. Tle 1. Sreening of Phytohemil onstituents S. No. Phytohemil onstituents Pet. ether extrt of Nelumo nuifer seed Ethnoli extrt of Nelumo nuifer seed 1. Alkloids Flvonoids Crohydrtes Proteins nd mino ids Sponins Tnnins Phenols Glyosides Polysterols Present; Asent Preliminry phytohemil investigtions (T. 1) of NNSEE hs showed positive results to lkloids, sponins, tnnins, flvonoids, rohydrtes, proteins nd mino ids. The presene of these iohemil onstituents my show etter therpeuti tion to renl firosis [13]. An ute toxiity study for NNSEE ws onduted s per OECD Guidelines 423 y using Wistr lino rts. Eh group of nimls treted with the NNSEE solution y the orl route did not result in signs or symptoms of ute toxiity or mortlity within 24 hours nd further period for 14 dys. The otined dt on toxiity studies of NNSEE (T. 2) did not show ny lethlity (LD 5 ) in rts weighing up to 2,mg/kg. Aording to the OECD guidelines, sfe dose rnge, i.e. 1/5 (4mg/kg.wt) s test-2 dose nd 1/1 (2mg/kg.wt) s test-1 dose, ws used for susequent studies [14]. Moreover, the seletion of the niml model my depend on the progression of the disese, sine some models develop renl firosis rpidly, e.g. ureterl ostrution model (1 2 weeks), while others tke severl weeks, e.g. doxoruiin, Anti-Thy1 or protein overlod indued nephropthy. Furthermore, reproduiility in the level of disese is n importnt riterion in hoosing the niml model. In the urrent study it ws noted tht UUO nd Doxoruiin-induing methods re used mostly s models for induing firoti tivity. Other methods re very omplited nd time-onsuming proesses for induing disese. Therefore, in this study, the two ovementioned methods of induing the disese were seleted nd investigted. Tle 2. Aute toxiity studies of NNSEE Groups Dose (mg/kg) No. of nimls I 5mg/kg 3 II 5mg/kg 3 III 3mg/kg 3 IV 2,mg/kg 3 Signs of toxiity No signs of toxiity No signs of toxiity No signs of toxiity No signs of toxiity Onset of toxiity Mortlity rte Durtion of study Nil Nil 14 dys Nil Nil 14 dys Nil Nil 14 dys Nil Nil 14 dys Unilterl Ureter Ostrution (UUO). This model is hrterized y tissue loss, trophy of tuulr epithelil ells nd development of tuulointerstitil inflmmtion

8 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of 73 nd firosis [15]. It resemles onditions seen in humn ostrutive nephropthy. In the UUO model, one ureter is ligted to lok the flow of urine from kidneys to the urinry ldder. The umulted urine in the kidneys tivtes renl tuulr ells, whih eventully leds to firosis in the ostruted kidney, wheres the ontrlterl kidney n, to some extent, regulte renl funtion. In this study, renl firosis ws well estlished within 3 dys. Anormlities of renl funtions in nephrotoxi rts indued y UUO re inresed retinine, BUN levels, derese in totl protein, glomerulr filtrtion rte levels, tuulr ell nerosis nd glomerulr hyliniztion, whih led to profiroti responses [16]. The rts were then treted with stndrd drugs (Pirfenidone 2mg/kg.wt, p.o) nd NNSEE t dose of 2 nd 4mg/kg.wt p.o in rt (18 2g). After tretment, the iohemil prmeters like serum retinine(grph 5), BUN level ttenutes (Grph 6), totl protein(grph 7) nd GFR levels were elevted (Grph 8) in UUO- indued renl firosis. This reveled tht the NNSEE test-2 (4mg/kg.wt) showed greter effet thn the test-1 (2mg/kg.wt), whih indites nti-firoti tivity nd is potently dose dependent. Doxoruiin or Adrimyin-indued nephropthy. This model is hrterized y the development of proteinuri, fol segmentl glomeruloslerosis nd tuulointerstitil firosis. It mimis the proteinuri ondition in ptients, nd is onsidered n experimentl nlogue of humn fol glomeruloslerosis. Doxoruiin is n nti-neoplsti gent nd uses nephrotoxiity through mny mehnisms, suh s y induing oxidtive stress nd y ffeting wter nd ure trnsporters in the renl medull. In the presented study, renl firosis ws well estlished within 1 2 months of single intrvenous injetion of doxoruiin (4mg/kg.wt). Doxoruiin plys mjor role in deteriortion of the glomerulr struture, proteinuri, tuulr nd interstitil inflmmtion, ulminting renl firosis in rts. Biohemil prmeters were evluted, e.g. serum retinine (Grph 1), BUN levels were elevted (Grph 2), while totl protein (Grph 3)[17] nd GFR levels were deresed (Grph 4); tuulr ell nerosis in doxoruiinindued renl firosis After two months, rts were treted with the stndrd drug (Pirfenidone 2mg/kg.wt, p.o) nd NNSEE t dose of 2 nd 4mg/kg.wt p.o. NNSEE meliorted ll the profiroti responses. After tretment, NNSEE nd pirfenidone reversed ll the mnifesttions of renl firosis. NNSEE test-2 (4mg/kg.wt) showed etter therpeuti effet thn the test-1 (2mg/kg.wt), whih indites nti-firoti tivity nd is potently dose dependent. Antioxidnts nturlly present in the ody ply role in preventing the development of free rdils nd inhiit the oxidtion of other moleules. Oxidtion is hemil retion tht n produe free rdils, leding to hin retions tht my dmge ells. Cells re proteted ginst oxidtive stress y n interting network of ntioxidnt enzymes [18]. Antioxidnt enzymes, e.g. SOD, CAT nd peroxidses, re the mjor enzymes whih prevent free rdils. SOD is present in lmost ll eroi ells nd in extrellulr fluids. In humns, SOD is present in ytosol nd mitohondrion, nd lso exists in extrellulr fluids. SOD tlyzes the rekdown of the superoxide nion (free rdil) into oxygen nd hydrogen peroxide [19]. CAT is minly present in peroxisomes whih tlyze the onversion Tle 3. Evlution of Serum retinine, BUN, Totl protein nd GFR levels in doxoruiin indued group S. No Goups Norml Sline (4ml/kg) Positive ontrol Doxoruiin (4 mg/kg) Stndrd Pirfenidone (2 mg/kg) Test-1 NNSEE (2 mg/kg) Test-2 NNSEE (4 mg/kg) Serum retinine (mg/dl) BUN level (mg/dl) Totl protein level (g/dl) GFR (ml/ min/1.73m 2 ).966± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.567 All vlues re expressed s men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Tle 4. Evlution of Serum retinine, BUN, Totl protein nd GFR levels in UUO indued group S. No. Groups Serum retinine (mg/dl) BUN level (mg/dl) Totl protein level (g/dl) GFR (ml/ min/1.73m 2 ) 1. Norml (4ml/kg).96± ± ± ± Positive ontrol (Doxoruiin 4mg/kg) Stndrd (Pirfenidone 2mg/kg) Test-1 (NNSEE 2mg/kg) Test-2 (NNSEE 4 mg/kg) 7.68± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.498 All vlues re expressed s men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test). Tle 5. Evlution of Invivo ntioxidnt levels (SOD nd CAT) S. No. Groups SOD (Units/mg protein) CAT (Units/mg protein) 1. Norml (Sline 4ml/kg) 35.1 ± ± Positive ontrol ± ± Stndrd (Pirfenidone 2mg/kg) ± ± Test-1 (NNSEE 2mg/kg) ± ± Test-2 (NNSEE 4 mg/kg) ± ± 2.35 All vlues re expressed s men ± SEM, (n=6). Sttistil signifine p<.5, p<.1, p<.1, ws ompred with positive ontrol group (One-wy ANOVA followed y Dunnett s multiple omprisons test) of hydrogen peroxide to oxygen nd wter y using iron or mngnese oftors [2]. Peroxidses lso tlyze the redution of hydrogen peroxides [21]. In the urrent study, the ntioxidnt tivity of Superoxide dismutse (SOD) nd Ctlse (CAT) ws estimted. Oxidtive stress is produed y doxoruiin whih indues glomerulr toxiity in rts. Redox yling of the quinone

9 74 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of funtionl group doxoruiin is proposed s the key ftor in doxoruiin nephrotoxiity. Retive oxygen speies (ROS) my initite degenertive sde y the oxidtion of ellulr thiols nd lipid memrne strutures. Doxoruiin hs een suggested to upregulte NADPH-oxidse (NOX), n importnt soure of ROS in the kidney [22]. The generted retive oxygen speies, suh s superoxide nd hydroxyl rdils, hve the potentil to use dmge to vrious intrellulr omponents. A defiieny of oxygen supply my destroy the tuulr ells nd glomerulr ells in kidney tht leds to one of the reson for renl firosis. SOD tivity (Grph 9) dereses in renl firosis-indued nimls due to exessive formtion of superoxide free rdils, nd lso the tivities of svenging enzyme CAT (Grph1) signifintly. UUO-indued nd doxoruiin-indued nimls showed signifint redution of Superoxide dismutse (SOD) nd Ctlse (CAT) enzymes, ompred to the norml group of rts. Elevted retive oxygen speies my prompt the disese. The indued nimls were treted with NNSEE t dose of 2 nd 4mg/kg.wt whih reversed ll these mnifesttions fter the experimentl period. This my e due to the presene of polyphenols nd lkloids in the NNSEE whih hve n ntioxidnt property nd nephroprotetive potentil in dose dependent mnner. The test-2 (4mg/kg) of NNSEE showed more potent therpeuti tivity thn the test-1 (2mg/kg). In the histopthology of kidney tissue, it ws lerly noted tht hyliniztion of glomeruli, degenertive hnges, tuulr dmge, vuoliztion nd nerosis took ple, whih ll led to renl firosis [23]. Rts were then treted with the NNSEE, s well s the stndrd drug Pirfenidone, whih meliorted ll the profiroti responses, the regenertion of glomerulr ells took ple, nd exess hylinlized glomeruli deresed. NNSEE of test-2 (4mg/kg.wt) showed etter therpeuti tivity thn the test-1 (2mg/kg.wt) in dose dependent mnner, whih my e due minly to the presene of lkloids nd polyphenols. Hene, the presented study shows tht NNSEE hs n nti-firoti property in kidney y ltering the profiroti responses, nd lso showed ntioxidnt property y inresing SOD nd CAT levels, whih my e due to the presene of onstituents (lkloids nd polyphenols) present in the Nelumo nuifer seed. These results suggest tht NNSEE ould e effetive in reversing the profiroti responses in renl firosis. The test-2 (4mg/kg) of NNSEE showed greter therpeuti effet thn the test-1 (2mg/kg). CONCLUSION This reserh on renl firosis using doxoruiin nd UUOinduing models strongly implies tht the over-prodution of hylinized glomeruli my e due to the tivtion of TGF-β, whih uses repeted tissue injury leding to renl firosis. NNSEE meliortes renl firosis y inhiiting the reruitment of firoytes into the kidney, nd influenes ntioxidnt nd nti-firoti tivity. This sustntites the therpeuti utility in renl firosis. The results otined suggest tht NNSEE might e potentil gent to e pplied in linis for preventing the progression of renl firosis in dose dependent mnner. Further studies re needed to investigte the role of NNSEE in other models of renl firosis, suh s sutotl nephretomy, renl ishemi, nd Anti-Thy1 ntiody for the ext iohemil onstituents responsile for nti-firoti tivity. Aknowledgement The uthors express their grtitude to the mngement of Krishn Tej Phrmy College, Chdlwd Ngr, Tirupti, Chittoor distrit, Andhr Prdesh, Indi, for supported in the performne of this study. REFERENCES 1. Hideki Yokoi, Suqwr, Mukoym, Mori, Mkino, Suqnmi, Nqe, Yht, Fujing, Tnk, Nko.Role of onnetive tissue growth ftor in profiroti tion of trnsforming growth ftor-β: A potentil trget for preventing renl firosis. Amerin Journl of Kidney Disese 211; 38(4) (suppl 1): Ptrii silv 215, Deth report of kidney filure linked to firosis. Aville from 3. 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10 Journl of Pre-Clinil nd Clinil Reserh, 217, Vol 11, No 1 R Rushendrn, Dr. V Jysnkr Reddy, T Bhrth Kumr, T Mmth, J Roj, T Roopvni. Evlution of nti-firoti tivity of ethnoli extrt of Rhee, Ho Zoon Che nd Knghw Kim. Peroxiredoxins: historil overview nd speultive preview of novel mehnisms nd emerging onepts in ell signling. Free rdil iology nd Mediine 25; 38(12): CsImreSzly, KtlinErdelyi, Gor Kokeny, EnikoLjtr, Mri Godo, CsRevesz, TmsKusr, Norert Kiss, Mrt Srkozy, Tms Csont, Tior Krens, Gor Szensi, Pl Pher nd Peter Hmr. Oxidtive or Nitrtive stress nd inflmmtion drive progression of doxoruiin indued renl firosis in rts s reveled y ompring norml nd firosis resistnt rt strin. Journl Pone 215; 1(6): Alton Frris nd Roert Colvin. Renl interstitil firosis: Mehnism nd evlution. Current Opinion in Nephrology nd Hypertension 212; 21(3):