Research Article Protective Effect of Short-Term Genistein Supplementation on the Early Stage in Diabetes-Induced Renal Damage

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1 Meditors of Inflmmtion Volume 2013, rticle ID , 14 pges Reserch rticle Protective Effect of Short-Term Genistein Supplementtion on the Erly Stge in Dibetes-Induced Renl Dmge Min Ju Kim nd Yunsook Lim Deprtment of Food nd Nutrition, Kyung Hee University, Seoul , Republic of Kore Correspondence should be ddressed to Yunsook Lim; Received 14 Jnury 2013; Revised 25 Mrch 2013; ccepted 27 Mrch 2013 cdemic Editor: Fábio Sntos Lir Copyright 2013 M. J. Kim nd Y. Lim. This is n open ccess rticle distributed under the Cretive Commons ttribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Hyperglycemi-induced oxidtive stress hs been concerned in the development of dibetic nephropthy (DN), which my cuse kidney dmge ssocited with inflmmtion nd fibrosis. This study hs been conducted to investigte the role of genistein supplementtion in n cute DN stte. Mice with FG levels more thn 250 mg/dl fter lloxn injection (single i.p., 150 mg/kg) were considered s dibetic. Dibetic mice (DM) were further subdivided ccording to their FG levels, medium-high FG (DMMH < 450 mg/dl) nd high FG (DMH; 450 mg/dl) nd were dministrted by n IG-93G diet supplemented with different doses of genistein (0, or 0.1%). fter 2 weeks tretment, the levels of kidney mlondildehyde (MD), blood ure nitrogen (UN), nd plsm cretinine nd lipid profiles, s well s oxidtive stress nd inflmmtion-relted mrkers, were mesured (P < 0.05). Genistein supplementtion improved levels of FG in the DMMH groups, but not in the DMH group, regrdless of the tretment dose. Moreover, the supplementtion ttenuted kidney oxidtive stress indicted by MD, UN, nd plsm cretinine. In ddition, genistein tretment decresed inflmmtory mrkers such s nucler fctor kpp (p65), phosphorylted inhibitory kpp lph, C-rective protein, monocyte chemotctic protein-1, cyclooxygense-2, nd tumor necrosis fctor-lph nd improved oxidtive stress mrkers (nucler-relted fctor E2, heme oxygense-1, glutthione peroxidse, nd superoxide dismutse isoforms) in tretment groups, regrdless of the genistein tretment dose. Furthermore, genistein supplementtion inhibited the fibrosis-relted mrkers (protein kinse C, protein kinse C-bet II, nd trnsforming growth fctor-bet I) in the DN stte. However, 0.1% genistein supplementtion in dibetes with high FG levels selectively showed preventive effect on kidney dmge. These results suggest tht genistein might be good protective substnce for DN through regultion of oxidtive stress nd inflmmtion. In prticulr, genistein is more efficient in dibetes ptients with medium-high blood glucose levels. Finlly, it is required to estblish the beneficil dosge of genistein ccording to blood glucose levels. 1. Introduction Dibetes mellitus (DM) is mjor endocrine-metbolic disorder tht is ssocited with chronic hyperglycemi by disturbnce in crbohydrte, protein, nd lipid metbolism. ccording to the WHO (World Helth Orgniztion), the world prevlence of dibetes hs been incresing explosively from 171 million in 2000 to n ssumed 366 million in 2030 [1]. s DM hve severe helth consequences, it gives rise to dibetic complictions including retinopthy, neuropthy, nd nephropthy. bout 20% 40% of dibetic ptients suffer from dibetic nephropthy (DN), which is chrcterized by end-stge renl disese [2].DN hs been implicted in severl mechnisms by hyperglycemi, which my simulte overproduction of rective oxygen species (ROS). ROS ply crucil role in genertion of oxidtive stress nd severl inflmmtory responses [3, 4] tht trigger cellulr dysfunction nd progression of kidney fibrosis. Indeed, the response my be upregulted by ROS-relted ctivtion of trnscription fctors nd their downstrem genes. This fct suggests tht the mechnism of severl trnscription fctors is implicted in hyperglycemi-medited expression of genes involved in DN [5]. Recently, it hs become incresingly cknowledged tht NFκ generlly works with other trnscription fctors [6, 7], such s nucler relted fctor E2 (Nrf2) [8]. DN condition is expected to bring out diverse synergistic effects t the trnscriptionl level [6]. NFκ induced by oxidtive stress is one of the most criticl trnscriptionl regultory fctorsthtcontroltheexpressionoflrgenumberofgenes involved in inflmmtory response, including cytokines,

2 2 Meditors of Inflmmtion chemokines, growth fctors, nd dhesion molecules [9]. It medites dmges in extrcellulr mtrix, glomerulosclerosis, nd renl filure, thus stimulting the development of DN. Recently, n increse in NFκ ctivtionhsbeen observed in DM ptients [10, 11] nd in DN nimls [12]. In contrst to the inflmmtory ction of NFκ, Nrf2 is responsible for the defense system ginst oxidtive stress [13, 14] nd inflmmtion [8] by regultion of phse II detoxifying enzymes nd redox-relted ntioxidnt proteins [15]. It is known tht ctivtion of Nrf2 nd upregultion of its downstrem ntioxidnt genes in hyperglycemic condition were found not only in the cultured cells, but in DN ptients [16]. Therefore, Nrf2 my contribute to the improvement of inflmmtory conditions such s DN. With the onset of ROS production in dibetic kidneys, fibrosis is stimulted by increses in oxidtive stress nd inflmmtion. Protein kinse C (PKC) is ssocited with phosphoryltion of serine/threonine residues in insulin receptors nd is generted due to the synthesis of dicylglycerol (DG) under the high intrcellulr concentrtion of glucose [17, 18]. In prticulr, PKC-βII, s one of the vrious isoforms of PKC, is well known to ccelerte the pthogenesis of hyperglycemic kidney injuries, nd it leds to insulin resistnce s well s to dysfunction of vrious cells through the reduction of insulin receptor substrte- (IRS-) 2 tyrosine phosphoryltion, resulting in defected insulin stimultion nd intrcellulr ccumultion of dicylglycerol in vrious orgns [19, 20]. Thus, excessive production of PKC-βII in dibetic kidneys my induce formtion of dvnced glyction end products (GE), s well s production of growth fctors, such s trnsforming growth fctor-β (TGF-β), connective tissue growth fctor (CTGF), nd vsculr endothelil growth fctor (VEGF) [21, 22]. Genistein, clss of phytoestrogens known s isoflvones, is mostly found in legumes. It hs ttrcted ttention becuse of its beneficil effects on prevention of metbolic disorders relted to crdiovsculr disese (CVD), obesity, cncer, nd dibetes [23 26]. Thus, genistein hs been extensively estblished s multifunctionl gent through enhncing the ntioxidnt defense system nd nti-inflmmtion response. Recently,studyfocusedontheprotectiveroleofgenistein on renl mlfunction in rts fed fructose rich diet, through the modultion of insulin resistnce-induced pthologicl pthwys [27]. Furthermore, Yun et l. hve noted tht high doses of genistein ( 5 μmol L 1 ) protected renl mesngil cells ginst hyperglycemic condition, which incresed fibrosis through induction of fibrosis relted genes, such s extrcellulr mtrix (ECM) nd TGF-β [28]. nother study hs shown tht genistein injections (10 mg/kg vi i.p.) reduced urinry TRs excretion nd renl gp91phox expression, s well s decresed production of inflmmtory mrkers, including p-erk, ICM-1, nd MCP-1, in DN mice [29]. However, the efficcy of genistein on the connection of complex responses ssocited with oxidtive stress nd inflmmtion in DN is very uncertin. Moreover, little reserchhsfocusedontheroleofgenisteininthedevelopment of DN in ccordnce with the degree of fsting blood glucose levels. In this study, we hypothesized tht shortterm genistein supplementtion protects ginst dibetic kidney dmge, depending on fsting blood glucose levels, through enhncement of hyperglycemi-induced oxidtive stress, inflmmtion, nd fibrosis in DN. 2. Mterils nd Methods 2.1. nimls. 5.5-week-old femle ICR mice were obtined from Dehn iolink Co., LTD (Eumseong, Choungcheongbuk-do, Republic of Kore). Mice were individully housed in cges nd cclimted for week in niml fcility conditions (22 ± 1 Cnd50 ± 1%humiditywith12hinthelight/drk). Dibetes ws induced with single intrperitonel (i.p.) injection of 150 mg/kg lloxn monohydrte (Sigm-ldrich Co., St Louis, MO, US) in sline. On the other hnd, nondibetic control mice were injected with only sline in the sme mnner s the dibetic mice were treted. fter 1- week tretment, the induction of dibetes ws confirmed by mesuring fsting blood glucose levels. Fsting blood glucose levels from the mouse til vein were mesured by using onetouch blood glucose meter (LifeScn Inc., Milpits, US). Fsting blood glucose levels 250 mg/dl were considered s dibetes. ll mice cre nd experiments were pproved bythenimlcreinstitutionlcommitteeofkyunghee University, Seoul, Republic of Kore Experimentl Design. Dibetic mice were subdivided into two groups in ccordnce with fsting blood glucose (FG) levels: medium high FG (DMMH; 250 mg/dl FG levels 450mg/dL)ndhighFG(DMH;450mg/dL FG levels 600 mg/dl). Mice were treted with different diets nd divided into the following groups (n =9-10 per group): non-dibetic mice () nd dibetic-control mice (DMC; DMMH-C, DMH-C) mice were fed n IN-93G diet without genistein supplementtion (0%). DM-0.025% (0.025% genistein; DMMH-0.025%, DMH-0.025%) mice were fed 0.025% genistein (LC Lbortories, Woburn, M) supplementtion. DM-0.1% (0.1% genistein; DMMH-0.1%, DMH-0.1%) mice were fed 0.1% genistein supplementtion. More detils re shown in Tble 1. t the end of the tretment (2 weeks), body weight, food consumption, nd fsting blood glucose levels were mesured once week. Mice were fsted 8 h nd nesthetized with isoflurne. lood smples were collected by crdic puncture, nd then they were centrifuged t 3000 rpm for 10 min t 4 C. The kidneys were wshed in sline nd frozen immeditely in liquid nitrogen. ll smples were stored t 80 C until subsequent nlysis Mesurement of Serum iochemicl nlysis (Lipid Profile). lood smples were collected in heprin pretretedtubes nd centrifuged t 3000 rpm for 15 min to obtin plsm. The concentrtions of totl cholesterol (TC), triglyceride (TG), nd high-density lipoprotein (HDL) cholesterol in plsm were ssyed using the enzymtic method. riefly, 20 μl of plsm ws mixed with n enzymtic kit (io- Clinicl System, Gyeonggi-do, Republic of Kore) nd incubted t 37 C wter bth for 10 min. Concentrtions were determined t 505 nm, 550 nm, nd 500 nm, respectively.

3 Meditors of Inflmmtion 3 Tble 1: Clssifiction of experimentl groups. Group Tretment Nondibetic mice were fed IN-93G diet without genistein supplementtion Dibetic-control mice with the level of medium-high FG between 250 nd 450 were DMMH-C fed IN-93G diet without genistein supplementtion Dibetic mice with medium high FG levels DMMH-0.025% between 250 nd 450 were fed 0.025% genistein supplementtion Dibetic mice with medium high FG levels DMMH-0.1% between 250 nd 450 were fed 0.1% genistein supplementtion Dibetic-control mice with the level of high DMH-C FG between 450 nd 600 did not receive genistein supplementtion Dibetic mice with high FG levels between DMH-0.025% 450 nd 600 were fed 0.025% genistein supplementtion Dibetic mice with high FG levels between DMH-0.1% 450nd600werefed0.1%genistein supplementtion The therogenic index (I) of plsm ws clculted by the following rtio: (TC/HDL-C)/HDL-C Renl Function Monitoring lood Ure Nitrogen (UN) Mesurement. Kidney function ws mesured by UN. Specimens were exmined by commercilly vilble kit (sn phrmceuticl, Seoul, Republic of Kore) nd incubted in 37 Cwterbth for 5 min. Then, concentrtions were determined t 580 nm using n ELIS reder (IO-TEK instruments, Winooski, VT, US) Plsm Cretinine. Plsm cretinine levels were exmined by cretinine ssy kit (io-clinicl System, Gyeonggi-do, Republic of Kore) ccording to the mnufcturer s protocol. riefly, mixture of plsm nd picric cid were centrifuged t 3000 rpm for 10 min. Superntnt ws rected by n NOH regent t room temperture for 20 min nd determined t 515 nm using n ELIS reder Mlondildehyde (MD) Mesurement in Kidneys. Mlondildehyde (MD) mesurement ws usully used for estimtion of lipid peroxidtion levels [30]. riefly, kidney homogentes were prepred in 0.15 M KCl buffer. totl of 200 μl of homogented kidney tissues were mixed with 200 μl of 8.1% SDS nd incubted t room temperture for 10 min. totl of 3 ml of 20% cetic cid-0.8% thiobrbituric cid (T) mixture (1 : 1, v/v) nd 600 μl ofdistilledwter were dded to mke totl volume of 4 ml. The mixture ws heted for 1 h in 95 Cwterbth.ftercoolingin ice wter, 1 ml distilled wter nd 5 ml mixture of n- butnol nd pyridine (15 : 1, v/v) were dded to ech tube. fter centrifuging t 4000 rpm for 10 min, the upper lyer ws mesured t 532 nm using n ELIS reder. Concentrtions were determined using 1,1,3,3-tetrmethoxypropne (TMP, sigm-ldrich, St. Louis, MO., US) s stndrd Preprtion of Western lot. For extrction of whole protein, 0.1 g of kidney tissues ws homogented t 4 C in lysis buffer (contining 20 mm Tris-HCl, 150 mm NCl, ph7.5, 1% NP40, 0.5% N-deoxycholte stock, 1 mm EDT, 0.1% SDS) with protese inhibitor (Sigm ldrich) nd centrifuged t 14,000 rpm for 30 min. The resulting superntnts were frozen t 80 C until western blot nlysis. Nucler extrcts were prepred from 0.25 g of kidney tissue nd homogented in 5 ml of buffer (0.6% NP40, 150 mm NCl, 10 mm HEPES (ph7.9), 1 mm EDT, 0.5 mm PMSF, Leupeptin, Pepsttin, nd protinin). fter centrifugtion (2,000 rpm, 4 C, 30 sec), the superntnts incubted on ice for 5 min, centrifuged t 5,000 rpm for 5 min, nd discrded the superntnt. 200 μl ofbuffer(25%glycerol,20mm HEPES (ph7.9), 420 mm NCl, 1.2 mm MgCl 2, 0.2mM EDT, 0.5 mm dithiothreitol (DTT), 0.5 mm PMSF, enzmidine, Leupeptin, Pepsttin, nd protinine) ws dded to the resulting pellet nd shcked on ice for 20 min. The resulting suspensions were frozen t 80 C until western blot nlysis. Protein concentrtion ws mesured using the NnoPhotometer (Implen, Germny). For gel electrophoresis, n equl mount of cytosolic nd nucler protein extrcts (50 μg nd25μg of totl protein) ws loded in ech lne. Proteins were seprted by 10% SDS-PGE nd then trnsferred to the PVDF membrne (Millipore, Mrlborogh, M, US). The membrne ws blocked with 5% nonft milk or 3% S in PS contining Tween 20 (PST) nd probed overnight t refrigertion temperture with primry ntibodies ginst Nrf2 (dilution 1 : 1000; bcm), HO-1 (dilution 1 : 1000; Stressgen), GPx (dilution 1 : 16000; bcm), CuZn- SOD (dilution 1 : 1000; Snt Cruz iotechnology), MnSOD (dilution 1 : 1000; Stressgen), p65 (dilution 1 : 200; Snt Cruz iotechnology), piκα (dilution 1 : 200; Snt Cruz iotechnology), TNF-α (dilution 1 : 200; Snt Cruz iotechnology), CRP (dilution 1 : 200; bcm), MCP-1(dilution 1 : 1000; Cell Signling), COX-2 (dilution 1 : 200; Stressgen), PKC (dilution 1 : 200; Snt Cruz iotechnology), PKC-βII (dilution 1 : 200; SntCruziotechnology),TGF-β1 (dilution 1 : 200; Snt Cruz iotechnology), nd β-ctin (dilution 1 :1000; Snt Cruz iotechnology). The membrne ws wshed with PST nd incubted with n HRP-conjugted secondry ntibody (Snt Cruz iotechnology, C, US) for 1 h. The trget proteins were detected nd visulized by enhnced chemiluminescence western blotting gents (Elpis iotech, Republic of Kore) on n Imge nlyzer (G box, Syngene, UK). The quntittion of ech protein expression compred to the βctin protein expression level ws performed Sttisticl nlysis. ll dt re presented s men ± SD. Smple normlity ws tested for primry outcomes (body weight, food intke, nd fting blood glucose level). Sttisticl differences of vribles (body weight, food intke, nd fsting blood glucose level) between nd DMH-C

4 4 Meditors of Inflmmtion 30 8 ody weight (g) Food intke (g) W0 W1 W2 Genestein tretment time (week) 0 W2 W1 Time fter tretment of diet (week) DMC DM-0.025% DM-0.1% () DMC DM-0.025% DM-0.1% (b) Figure 1: Effect of genistein supplementtion on body weight () nd food intke (b) in experimentl mice. Dt re presented s mens ± SD (n = 9-10/group). Men vlues with different letters were significntly different, P < Sttisticl differences of vribles between nd DMH-C nlyzed by unpired t-test were shown in cpitl letters., control mice; DMC, dibetic control mice; DM-0.025%, dibetic mice supplemented with 0.025% genistein; DM-0.1%, dibetic mice supplemented with 0.1% genistein. were nlyzed by unpired t-test. The effects of DM severity (norml control, DMMH, nd DMH) nd/or genistein supplemented diet (0, 0.025, nd 0.1%) were nlyzed by one-wy nlysis of vrince (NOV). Two-wy NOV ws used to nlyze the effects of the genistein supplemented diet nd DM severity nd their interction on outcomes followed by post hoc test (Tukey HSD) using SPSS (20.0 K for Windows) sttisticl nlysis progrm. For ll outcomes, vlue of P< 0.05 ws considered significnt. 3. Results 3.1. Effect of Genistein Supplementtion on ody Weight nd Food Intke. Chnges in body weight nd food intke during the experimentl period re shown in Figure 1. fter 1 week of lloxn injection to induce dibetes, body weight in dibetic mice ws significntly lower thn tht of (Figure 1()). However, genistein supplementtion, regrdless of dose, did not prevent the decrese in body weight. Food intke ws significntly incresed in dibetic mice, regrdless of genistein supplementtion compred with the group (Figure 1(b)) Effect of Genistein Supplementtion on Chnges in Fsting Glucose Level. Levels of fsting blood glucose were significntly higher in ll the dibetic groups compred to the group. The 0.025% genistein supplementtion in DMMH significntly decresed FG levels, but 0.1% genistein in DMMH did not significntly reduce FG levels (Figure 2()). In Figure 2(b), genistein supplementtion in DMH did not show difference in FG levels Effect of Genistein Supplementtion on iochemicl Mrkers Lipid Profiles. To exmine the effect of genistein supplementtion on lipid profiles, we mesured plsm lipid profiles. s shown in Tble 2(), plsm levels of totl cholesterol (TC) nd triglycerides (TG) were elevted in the DMC more thn in the, but there were no significnt differences between the DMC groups nd genistein supplementtion groups. Moreover, the plsm level of high density lipoprotein cholesterol (HDL-C) did not differ mong the groups. Thus, the DMC groups were chrcterized by mrkedly elevted therogenic index (I) s compred to the group, but genistein supplementtion did not effectively decrese I lood Ure Nitrogen (UN). s shown in Tble 2(b), the concentrtion of UN ws significntly incresed in the DMMH-C nd DMH-C groups compred to tht of the group (P < 0.05). UN concentrtions of 0.025% DMMH nd 0.1% DMMH were decresed by 47% nd 43%, respectively, s compred to the DMMH-C group, UN concentrtions of 0.025% DMH nd 0.1% DMH groups were significntly decresed by 52% nd 51%, respectively, s compred to the DMH-C group Plsm Cretinine. s shown in Tble 2(c), plsm cretinine levels were significntly elevted in the DMMH- C nd the DMH-C groups compred with the group (P < 0.05). The concentrtion of plsm cretinine ws muchhigherinthedmh-cgroupthninthedmmh-c group. Genistein supplementtion, regrdless of dose, in the DMMH group meliorted plsm cretinine levels. However, lthough the plsm cretinine level of the DMH group

5 Meditors of Inflmmtion Fsting glucose levels (mg/dl) b b, b b, Fsting glucose levels (mg/dl) W0 W1 W2 Genistein tretment period (week) 0 W0 W1 W2 Genistein tretment period (week) DMMH-C DMMH-0.025% DMMH-0.1% DMH-C DMH-0.025% DMH-0.1% () (b) Figure 2: Effect of genistein supplementtion on fsting blood glucose levels in experimentl mice. Fsting blood glucose levels in DMMH group()ndfstingbloodglucoselevelsindmhgroup(b).dtrepresentedsmens± SD (n =9-10/group). Men vlues with different letters were significntly different, P < Sttisticl differences of vribles between nd DMH-C nlyzed by unpired t-test were shown in cpitl letters nd effects of the genistein supplemented diets on body weight nd food intke in dibetic mice using one-wy NOV ws represented by smll letters. Group TC (mg/dl) Tble 2: Effect of genistein supplementtion on biochemicl mrkers. Lipid profiles Kidney function Oxidtive stress TG HDL-C UN Cretinine MD I (mg/dl) (mg/dl) (mg/dl) (mg/dl) (nm) ± ± ± ± ± ± ± 4.28 DMMH-C ± ± ± ± ± Cb 0.81 ± 0.13 b ± 6.23 b DMMH-0.025% ± ± ± ± ± ± ± 3.01 DMMH-0.1% ± ± ± ± ± ± ± 2.97 DMH-C ± ± ± ± ± Cb 0.94 ± 0.25 Cc ± 3.57 b DMH-0.025% ± ± ± ± ± ± 0.11 b ± 5.26 DMH-0.1% ± ± ± ± ± ± 0.21 bc ± 4.94 b bbrevitions: TC: totl cholesterol, TG: triglyceride, HDL: high density lipoprotein cholesterol, I: therogenic index, UN: blood ure nitrogen, nd MD: mlondildehyde. Men vlues with different letters were significntly different (P < 0.05). Sttisticl differences of vribles mong, DMMH-C, nd DMH-C nlyzed by one-wy NOV were shown in cpitl letters nd effects of the genistein supplemented diet nd/or DM severity using two-wy NOV were represented by smll letters. rechedmorethn1.5-foldcompredtothegroup,only the 0.025% genistein supplementtion significntly decresed plsm cretinine levels in DMH MD. To exmine the effect of genistein supplementtion on oxidtive stress in kidneys, kidney MD levels were mesured. MD levels were significntly elevted in both the DMMH-C nd DMH-C groups (1.5-fold bove, P < 0.05). On the other hnd, genistein supplementtion in the DMMH-C groups reduced the level of MD concentrtion to the norml level. The supplementtion of 0.025% genistein significntly decresed the kidney MD levels (DMMH-L; 33.26%, DMH-H; 29.22% compred to DMC), but the supplementtion of 0.1% genistein did not significntly reduce it in the DMH mice (Tble 2(d)) Effect of Genistein on Protein Expression Levels of Oxidtive Stress Mrkers in Dibetic Kidneys. We performed western blot nlysis to determine whether genistein supplementtion declined the ctivtion of Nrf2-linked oxidtive stress proteins in DN. The levels of cytosolic Nrf2 protein expression decresed in the DMMH-C nd DMH-C groups (P < 0.05, Figure 3()). We found tht the reduction of cytosolic Nrf2 protein levels in DMMH ws effectively restored by genistein supplementtion regrdless of dose. The 0.025% genistein supplementtion in DMH significntly rised the cytosolic Nrf2 levels, more thn the DMH-C (P < 0.05), but 0.1% genistein in DMH did not significntly ffect cytosolic Nrf2 expression. The expression of HO-1 levels, representtive trget gene in the Nrf2 pthwy, ws significntly incresed in the DMC s compred with the (P < 0.05). In ddition, the expression of

6 6 Meditors of Inflmmtion (.u.) Nrf2-relted mrkers C D E C, b b, b, b, b, b b 0.0 Cytosolic Nrf2 HO-1 GPx CuZnSOD MnSOD DMMH-C DMMH-0.025% DMMH-0.1% DMH-C DMH-0.025% DMH-0.1% Cytosolic Nrf2 HO-1 GPx CuZnSOD MnSOD β-ctin Figure 3: Effect of genistein supplementtion on the kidney protein levels of cytosolic Nrf2 (), HO-1 (b), GPx (c), CuZnSOD (d), nd MnSOD (e) in experimentl mice. ll results were conducted t lest three times. Dt re presented s mens ± SD (n =9-10/group). Men vlues with different letters were significntly different, P < Sttisticl differences of vribles mong, DMMH-C, nd DMH-C nlyzed by one-wy NOV were shown in cpitl letters nd effects of the genistein supplemented diet nd/or DM severity using two-wy NOV were represented by smll letters. HO-1 ws much higher in the DMC-C group thn in the DMMH-C group. Genistein supplementtion, regrdless of dose, completely reduced the expression of HO-1 levels in DMMH nd DMH (Figure 3(b)). Furthermore, GPx levels were significntly incresed in DMC mice, more so thn in mice (Figure 3(c)). GPx expression in the DMMH group ws normlized by genistein supplementtion independently of the dose. In the DMH group, 0.025% genistein supplementtion significntly reduced GPx expression, while 0.1% genistein supplementtion ws not chnged. s shown in Figure3(d), the expression of CuZnSOD levels ws higher in the DMMH-C nd the DMH-C thn in the. Genistein supplementtion reltively decresed the CuZnSOD levels, lthough the difference ws not sttisticlly significnt. Unfortuntely, the expression of MnSOD levels did not significntly differ mong the groups (Figure 3(e)) Effect of Genistein on Protein Expression Levels of Inflmmtion Mrkers in Dibetic Kidneys. We tested to elucidte whether the genistein supplementtion reduced the expression of NFκ-relted inflmmtory proteins in DN. The levels of NFκ (p65) nd piκα, n indirect mrker formesuringthectivtionofnfκ, were significntly incresedinthedmcscompredtothe(figures4() nd 4(b)). Genistein supplementtion, regrdless of dose, significntly decresed the levels of cytosolic piκα nd nucler NFκ in DMMH nd DMH compred to DMMH- C nd DMH-C(P < 0.05). Next, we mesured the expression of CRP, which ws incresed by lloxn-induced dibetes (Figure 4(c)). Genistein supplementtion, regrdless of dose, significntly inhibited incresed CRP levels in the DMMH nd DMH groups (P < 0.05). s shown in Figure 4(d), MCP-1levelswerehigherinDMMH-CndDMMH-Cthn in, nd the levels in DMMH nd DMH were mrkedly lower by 0.025% genistein. However, 0.1% genistein showed no significnt inhibitory effects on the MCP-1 levels in DMMH nd DMH. The protein expression of COX-2, s representtive mrker of the NFκ-pthwy, ws significntly

7 Meditors of Inflmmtion NFκ-relted mrkers C D E C, c, b, b F (.u.) 2 1, b b C, b, b b, b, b b b, b, b, b b 0 Nucler p65 Cytosolic piκα CRP MCP-1 COX-2 TNF-α DMMH DMMH-0.025% DMMH-0.1% DMH DMH-0.025% DMH-0.1% Cytosolic piκα CRP Nucler p65 β-ction MCP-1 COX-2 TNF-α β-ctin Figure 4: Effect of genistein supplementtion on the kidney protein levels of p65 (NFκ) (), piκα (b), CRP (c), MCP-1 (d), COX-2 (e), nd TNF-α (f) in experimentl mice. ll results were conducted t lest three times. Dt re presented s mens ± SD (n =9-10/group). Men vlues with different letters were significntly different, P < Sttisticl differences of vribles mong, DMMH-C, nd DMH-C nlyzed by one-wy NOV were shown in cpitl letters nd effects of the genistein supplemented diet nd/or DM severity using two-wy NOV were represented by smll letters. elevted in DMC (P < 0.05). Genistein supplementtion in DMMH suppressed the upregultion of COX-2 levels, while there ws no difference in the DMH groups (Figure 4(e)). dditionlly, TNF-α levels were significntly higher in ll dibetic mice thn in mice (Figure 5(f)). However, the genistein supplementtion groups exhibited remrkble reduction in the expression of TNF-α in comprisonwith the DMC groups, excluding DMH-0.1% (P < 0.05) Effect of Genistein on Protein Expression Levels of Fibrosis- Medited Mrkers in Dibetic Kidneys. We exmined the question s to whether genistein supplementtion contributed to enhncing n ntidibetic kidney fibrosis pthwy in the experimentl mice. Our dt showed significnt increse in PKC nd PKC-βII protein expression in the DMC groups, regrdless of FG levels (Figures 5() nd 5(b)). The levels of PKC nd PKC-βII protein expression in DMMH were effectively decresed by genistein supplementtion regrdless of dose (P < 0.05). The level of PKC expression in DMH ws reduced by genistein supplementtion, regrdless of dose, but there ws not significnt difference (Figure 5()). The 0.025% genistein supplementtion in DMH ws more effective t reducing the level of PKC-βII protein expression thn the 0.1% genistein in DMH (Figure 5(b)). To further investigte the mechnism of n ntifibrosis effect of genistein, we tested the expression of TFG-βI. TFG-β1, s one of the most potent fibrogenic response mrkers, ws greter in DMC thn those of. However, s contrsted with nontreted genistein supplementtion, genistein supplementtion groups experienced significnt decresed expression of TFG-βI, except DMH-0.1% (P < 0.05). 4. Discussion The present study provides some good evidence tht genistein hs n bility to protect kidneys from hyperglycemi-induced oxidtive stress, inflmmtion, nd fibrosis in lloxninduced dibetic mice. lthough genistein hs beneficil influences with respect to both ntioxidtive stress nd ntiinflmmtion [31], there is no cler underlying mechnism by

8 8 Meditors of Inflmmtion (.u.) , b Prefibrosis-relted mrkers, b, b b, b C, b b PKC PKC-βII TGF-βI DMMH DMMH-0.025% DMMH-0.1% DMH DMH-0.025% DMH-0.1% PKC PKC-βII TGF-βI Figure 5: Effect of genistein supplementtion on the kidney protein levels of PKC (), PKC-βII (b), nd TFG-βI (c) in experimentl mice. ll results were conducted t lest three times. Dt re presented s mens ± SD (n =9-10/group). Men vlues with different letters were significntly different, P < Sttisticl differences of vribles mong, DMMH-C nd DMH-C nlyzed by one-wy NOV were shown in cpitl letters nd effects of the genistein supplemented diet nd/or DM severity using two-wy NOV ws represented by smll letters. β-ctin whichgenisteincnboostprotectiveroleindnprogression in ccordnce with blood glucose levels. severe loss of body weight (.W.) nd n increse of food intke generlly occur in dibetic conditions [32 34]. We lso exmined the question s to whether genistein supplementtion did not improve body weight loss nd food intke s shown in the previous studies [35, 36]. n ultimte tretment gol of dibetes nd its complictions is the control of the FG levels[37]. ccording to previous studies, glucose level with mg/dl ws dignosed s mild hyperglycemi [38],nd glucose level bove 450 mg/dl ws considered s severe hyperglycemi [39]. In this study, dibetes with different FG levels, in rnge from 250 mg/dl to 600 mg/dl (mximum red by commercil glucometer), nd FG levels of 450 mg/dl were used s the criteri of hyperglycemi clssifiction. Our dt found tht genistein supplementtion decresed the FG level in DMMH mice, but it did not ffect blood glucose levels in DMH mice. Previously, genistein hs been shown to hve n effect on the modultion of blood glucose levels in vivo, regrdless of the mnner of genistein dministrtion nd tretment period nd dose, which hve included short-term (for 16 dys) i.p. injection of genistein (1 mg/kg.w./dy) in rts fed fructose rich diet [27] nd long-term (for 9 weeks) dietry supplementtion of genistein (0.02% w/w) in nonobese dibetic (NOD) mice [35]. In other findings [40], it ws discovered tht not low dose (under 15 mg/kg.w.) but high dose (15 30 mg/kg.w.) of genistein supplementtion mrkedly reduced blood glucose levels in lloxn-induced dibetes mice. However, reserchers hve not proven potentil benefit of genistein on dibetic nimls with different levels of FG. Collectively, the results suggested tht shortterm supplementtion of genistein possesses the cpcity to reduce hyperglycemi in the DMMH group without insulin tretment but not in DMH group. Impirment of insulin secretion in dibetes increses the relese of free ftty cids (FF) into the liver, nd it my cusenincreseintriglycerideproduction[41]. It promotes dibetic dyslipidemi, which my worsen the interply of inflmmtion nd intrrenl fibrosis [42, 43]. previous study reported tht genistein supplementtion (600 mg/kg diet) for 3 weeks improved plsm lipid profiles (TC, TG, nd HDL) in dibetic mice [44], wheres nother study confirmed tht genistein supplementtion (250 mg/kg diet) for 4 weeks did not improve the plsm lipid profiles in dibetic mice [45]. Our dt showed tht genistein supplementtion, regrdless of supplementtion doses (0.025% in 250 mg/kg diet or 0.1% in 1000 mg/kg diet), did not show lowering effect on dyslipidemi. These results suggest tht dibetes-relted dyslipidemi is controlled by reltively high concentrtion of genistein supplementtion for longer thn 3 weeks of tretment. UN nd plsm cretinine, s wste products of metbolism, prennounce dmge in kidney function [46].

9 Meditors of Inflmmtion 9 We observed tht UN nd plsm cretinine levels were incresed in DMC mice, especilly in DMH-C. Sung et l. [47] reported tht the genistein ddition (10 mg/kg.w.) for 3 dys significntly reduced UN nd serum cretinine levels in cispltin-induced cute renl injury. We lso observed tht genistein supplementtion decresed UN levels in DMMH group nd DMH group. UN is usully done together with plsm cretinine, which is more sensitive mrker of kidney dmge. Genistein supplementtion in DMMH llevited plsm cretinine levels to norml levels nd significntly reducedthelevelsindmh-0.025%,butnotindmh-0.1%. Thus, our dt demonstrted tht genistein, regrdless of supplemented dose, could prevent n impirment of kidney function in DMMH, nd only the 0.025% genistein supplementtion my hve beneficil effects on kidney dmge when the FG level is very high. In dibetic conditions, continuous overproduction of ROS nd n ntioxidnt defense system my cuse mitochondril impirment [48]. Thus, oxidtive stress is considered s meditor in tissue injury, including liver, brin, nd kidney. The kidney is known s highly sensitive orgn in oxidtive stress conditions becuse lipid composition in kidneys comprises long-chin polyunsturted ftty cids [49]. In our experiments, the MD ccumultion ws incresed by consequences of oxidtive stress, such s dibetes [50], but genistein (6 mg/kg/.w.) decresed the MD levels in the brinndliverofstz-induceddibeticmice[51]. Our study lso observed tht genistein supplementtion significntly lowered kidney MD levels in dibetic mice, except in DMH- 0.1%. previous study demonstrted tht high dose of genistein cn hve dverse ctions s prooxidnt, depending on the sttus of oxidtive stress [52]. Therefore, the results suggest tht 0.1% genistein supplementtion my ct s prooxidntinthedmhgroup,whichisconsidereds possessing higher oxidtive stress sttus compred to the DMMH group. Nrf2 is normlly combined with its repressive protein Kep1 (Kelch-like ECH-ssocited protein-1) in cytoplsm [53]. In n oxidtive stress stte, Nrf2 is seprted from Kep1 nd trnslocted to the nucleus. It ctivtes ntioxidnt enzymes such s HO-1, GST, NDH(H) quinoline oxidoreductse-1 (NQO1), nd glutthione peroxidse (GSH- Px) [54, 55]. Therefore, Nrf2 nd its downstrem genes ply crucil role in defense of cellulr dmge ginst oxidtive stress, but its overproduction my led to prdoxicl effects in connection with disturbnce in the protection of cells from oxidtive dmge [56]. Previous studies reported tht expression of Nrf2 nd ntioxidnt genes, such s HO-1, SOD, ctlse (CT), nd GPx, ws incresed in dibetes [57 59]. The results suggest tht excessive production of oxidtive stress seems to stimulte increses in ntioxidnt enzyme production in order to eliminte oxidtive stress gents in DM. It is known tht genistein hs cytoprotective effects on Nrf2 ctivtion nd its downstrem ntioxidnt enzymes, including HO-1, SOD, CT, nd GSH [60]. Our dt showed tht cytosolic Nrf2 ws decresed in dibetes, which my led to n increse in nucler Nrf2 ctivtion s consequence of the ctivtion of cellulr ntioxidnt defense with incresed trnscription of ntioxidnt genes. The results were reversed by the genistein supplementtion, nd this outcome supports the hypothesis tht genistein supplementtion ws ble to reestblish the cell homeostsis. However, 0.1% genistein in DMH did not mrkedly chnge Nrf2 levels. These findings indicte tht 0.1% genistein my be not enough to provide beneficil effects on the Nrf2-medited oxidtive stress pthwyindibeticmicewithhighfglevels. HO-1,representtivemrkerofnNrf2-reltedstress response, hs been found to increse in pthologicl conditions such s dibetes [61 63]. The present study demonstrted tht genistein supplementtion, regrdless of dose, tendstoreducetheexpressionofho-1levelsindmmhnd DMH. Moreover, protein levels of GPx nd SOD isoforms re ssocited with oxidtive dmge nd mitochondril dysfunction through hydrogen peroxide (H 2 O 2 )production by the glucose oxidse system [64 66]. previous study [67] demonstrted tht GPx ctivity ws incresed in dibetic mice orgns including the liver, pncres, nd kidney. Our findings proved tht genistein supplementtion reduced GPx levels, except the DMH-0.1% group. However, genistein did not significntly reduce CuZnSOD levels nd did not chnge MnSOD levels mong the groups. These results suggest tht genistein supplementtion selectively llevited oxidtive stress through the regultion of Nrf2 levels nd its consequent events. Moreover, the DMH group with high dose supplementtion of genistein my hve more oxidtive stress. Nrf2-medited interply hs two sides of ction s either regultor of ntioxidnt response or rective promoter of oxidtive stress in bnorml conditions [68]. On the bsis of our results, we proposed tht n overproduction of rective oxygen species (ROS) in dibetes cn trigger ctivtion of nucleus Nrf2 nd trnscription of its downstrem trget enzymes. On the other hnd, oxidtive stress leds to ctivtion of the inflmmtory-medited trnscription fctor, NFκ. Thus, we identified the fct tht genistein supplementtion ttenuted the hyperglycemi-induced inflmmtory responses through the regultion of the NFκ pthwy. Mny studies hve reported experimentl evidence showing tht NFκ ws ctivted in dibetic kidneys [69, 70], nd genistein supplementtion (1 mg/kg/.w.) ttenutes NFκ (P65) ctivtion in kidneys of rts fed fructose rich diet [71]. We hve investigted the piκα level in cytosol nd NFκ (p65) level in nucleus to identify NFκ ctivtion. piκα level is representtive of NFκ ctivtionincytosol becuse piκα fter phosphoryltion of Iκα is subsequently ubiquitinted nd degrded vi the protesome pthwy [72]. NFκ, p65 nd p50 heterodimer, seprted from Iκα is trnslocted into the nucleus nd ctivtes the expression of inflmmtory genes. Our dt showed tht the protein levels of piκα in cytosol nd NFκ innucleussincresed in DMC nd lowered in genistein supplementtion. These results imply tht genistein supplementtion blocked NFκ ctivtion by reduction of piκα. ctivtion of the NFκ signlingpthwyisknownto enhnce inflmmtory cytokines (IL-1β,TNF-α)nd ctivte fibrosis mrkers (GE, RGE) in dibetic mice [73]. mong them, TNF-α (tumor necrosis fctor-α) is the min proinflmmtory cytokine, which cts towrd the progression of dibetic kidney disese through recruitment of mcrophges

10 10 Meditors of Inflmmtion nd neutrophils into the kidney [74]. n incresed renl TNF-α level is correlted with indictors of renl filure in DM nimls [75] nd ptients[76]. The present study confirmed tht TNF-α levels in genistein supplementtion groups were even lower thn those in DMC groups. However, DMH with 0.1% genistein did not show significnt differences, which mens tht 0.025% genistein is more effective thn 0.1% genistein for DN with high FG levels. CRP is generlly incresed in inflmmtory conditions, such s those found in DN ptients nd nimls [77, 78]. Dietry isoflvone, including genistein, hs cpcity to decrese the concentrtion of CRP in humn plsm [79, 80]. Similrly, the expression of CRP levels in DN mice ws significntly reduced in ll dibetic mice supplemented with genistein, more so thn those of DMC. In ddition, expression of MCP-1 nd COX-2 is relevnt to NFκ-medited modultion of n inflmmtory cscde, which contributes to endothelil dysfunction [81 83]. Genistein (10 mg/kg vi i.p., three times week) s tyrosine kinse inhibitor hs been shown to reduce significntly the excretion of urinry MCP-1 in STZ-induced dibetic mice [29]. Our results showed tht the production of MCP-1 significntly decresed in the 0.025% genistein supplementtion groups,wheresthe0.1%genisteinsupplementtiongroups did not reduce MCP-1 production. Thus, the results suggest tht reltively low dose of genistein my reduce MCP-1 protein vi inhibitionofnfκ ctivtion. Moreover, genistein, s n inhibition gent of cell prolifertion, inhibited COX-2 protein in cncer cells [84], result tht improved the blnce of ngiogenesis nd poptosis. In our findings, overproduction of COX-2 in DMMH ws ttenuted by genistein supplementtion t both 0.025% nd 0.1% levels, but not in DMH. In other words, 0.025% genistein supplementtion in dibetes with medium high FG my control vsculr homeostsis through suppression of NFκ-medited inflmmtion. Moreover, the findings indicte tht Nrf2 ctivtion nd its downstrem signlling pthwy interct with the ctivtion of NFκ-medited inflmmtory responses in dibetes, nd genistein supplementtion might reduce ctivtion of ntioxidnt defence systems nd inflmmtory responses by regultion of Nrf2 nd NFκ interctions. Nrf2 nd NFκ interctions my ply serious role in fibrosis in dibetic kidneys, which corresponds with incresed PKC-medited pthwys in hyperglycemic conditions. This conclusion hs been supported by severl in vitro experiments [85], which demonstrted tht genistein (40 μm) blocked PKC ctivtion in VEGF-stimulted endothelil cells [86]. However, there is no reserch focusing on the effect of genistein on PKC inhibition in dibetic nimls. The PKC-β isoform is minly responsible for hyperglycemi-induced fibrosis in DN [87]. Severl reports hve provided evidence tht genistein ttenuted the levels of PKC isoenzymes, such s PKC-βI, in rt ventriculr monocytes [88], s well s levels of PKC-βII in rts fed fructose rich diet, n experiment tht constitutes hypertension mouse model [89]. Our dt showed the different effects of genistein on prefibrosis-relted mrkers, both PKC nd PKC-βII, in DMH depending on their tretment dose. The 0.1% genistein supplementtion in the DMH group did not significntly reduce the levels of both PKC nd PKC-βII. This result suggests tht 0.1% genistein supplementtion my not hve beneficil effects on fibrosis in dibetes with high FG. Continuous exposure of ROS in hyperglycemi my lso led to chnges in cell membrne structure. The trnsforming growth fctor βi (TFG-βI), fmily of fibrogenic cytokine, hs been generlly known to induce deposition of mtrix components,suchsecm,swellssynthesisofglomerulosclerosis in DN rts [90]. hyperglycemic condition inducesnincreseintgf-βilevels,nditstimultesfibrosis of numerous orgns, such s the kidney [91]. Thus, inhibition of TGF- βi iskeyplyerinprotectionofdibetickidneys. Genistein hs been proven effective in the prevention of hyperglycemi-induced fibrosis by inhibiting the expression of TGF- βi [28] ndtgf-βii [92]. In prticulr, the dt showed tht genistein ws ble to inhibit TGF-βI production, not t low concentrtion ( 5 μmol L 1 ),butthigh concentrtion ( 5 μmol L 1 )[28], nd to reduce TGF-βII production t the high concentrtion level (5 μg/ml) [92]. However, our results demonstrted tht 0.1% genistein supplementtion did not protect ginst the fibrosis process, represented by TGF-βII, from severe hyperglycemic condition in DMH. The results might be ssocited with the prooxidnt effect of genistein t high doses on severe hyperglycemi s promotor of prefibrosis in DN. Tken together, our dt evidenced tht genistein supplementtion inhibited hyperglycemi-induced fibrosis pthwys s well s the ctivtion of the trnscription fctors, Nrf2 nd NFκ. Moreover, we found tht genistein supplementtion hs selective effects on dibetic kidney dmge in ccordnce with FG levels. In previous studies, genistein hs been showntohvedverseeffectsinpthogeneticconditions, which my ct s prooxidnts nd ccelerte the progression of disese [93]. However, this study hs severl limittions. Only the short-term effects of dietry genistein supplementtion hve been investigted with respect to dibetes induced kidney dmge. Long-term supplementtion protocols my be helpful to verify the role of genistein in the DMH group (>450 mg/dl) becuse the DMH group my need longer time to control inflmmtion, oxidtive stress, nd fibrosis processes. Moreover, short-term supplementtion t different doses did not chnge plsm lipid profiles. This result might be ssocited with supplemented doses nd periods, s well s FG levels. In ddition to tretment regimens, histologicl nlysis of kidneys my be more helpful to investigte fibrosis process in this study. In conclusion, understnding the moleculr mechnisms tht regulte oxidtive stress, inflmmtion nd fibrosis is criticl not only in dibetic kidney dmge, but lso in other dibetic complictions. Hence, the results of this study my provide criticl insight into future nutritionl intervention strtegies, with or without insulin tretment, designed to prevent dibetic complictions ccording to FG levels. bbrevitions DN: Dibetic nephropthy FG: Fsting blood glucose ROS: Rective oxygen species

11 Meditors of Inflmmtion 11 UN: lood ure nitrogen NFκ : Nucler fctor kpp Nrf2: Nucler relted fctor E2 HO-1: Heme oxygense-1 CRP: C-rective protein MCP-1: Monocyte chemotctic protein-1 COX-2: Cyclooxygense-2 GPx: Glutthione peroxidse MD: Mlondildehyde CuZnSOD: Copper zinc superoxide dismutse MnSOD: Mngnese superoxide dismutse piκα: Phosphorylted inhibitory kpp lph TNF-α: Tumor necrosis fctor-lph PKC: Protein kinse C PKC-βII: Protein kinse C-bet II TGF-βI: Trnsforming growth fctor-bet I. Conflict of Interests The uthors nd mnufcturers disclose no ctul potentil conflictofinterests. cknowledgments This reserch ws supported by sic Science Reserch Progrm through the Ntionl Reserch Foundtion of Kore (NRF) funded by the Ministry of Eduction, Science nd Technology (2012-R ). References [1] C. Setcci, G. De Donto, F. Setcci, nd E. Chisci, Dibetic ptients: epidemiology nd globl impct, JournlofCrdiovsculr Surgery, vol. 50, no. 3, pp , [2] V. Vllon nd S. C. 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15 MEDITORS of INFLMMTION The Scientific World Journl Gstroenterology Reserch nd Prctice Journl of Dibetes Reserch Interntionl Journl of Journl of Endocrinology Immunology Reserch Disese Mrkers Submit your mnuscripts t iomed Reserch Interntionl PPR Reserch Journl of Obesity Journl of Ophthlmology Evidence-sed Complementry nd lterntive Medicine Stem Cells Interntionl Journl of Oncology Prkinson s Disese Computtionl nd Mthemticl Methods in Medicine IDS ehviourl Neurology Reserch nd Tretment Oxidtive Medicine nd Cellulr Longevity