Inhibition of ceramide redox signaling pathway blocks glomerular injury in hyperhomocysteinemic rats

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1 originl rticle & 6 Interntionl Society of Nephrology Inhiition of cermide redox signling pthwy locks glomerulr injury in hyperhomocysteinemic rts FYi 1, AY Zhng 1,NLi 2, RW Muh 2, M Fillet 3, A-F Renert 3 nd P-L Li 1,2 1 Deprtment of Phrmcology nd Toxicology, Medicl College of Wisconsin, Milwukee, Wisconsin, USA; 2 Deprtment of Phrmcology nd Toxicology, Medicl College of Virgini, Virgini Commonwelth University, Richmond, Virgini, USA nd 3 Lortory of Drug Anlysis, University of Liège, Liège, Belgium Cermide-ctivted NAD(P)H oxidse hs een reported to prticipte in homocysteine (Hcys)-induced norml metolism of the extrcellulr mtrix (ECM) in rt glomerulr mesngil cells. However, it remins unknown whether this cermide redox signling pthwy contriutes to glomerulr injury induced y hyperhomocysteinemi (hhcys) in vivo. The present study ws designed to ddress this question, defining the role of cermide nd ctivted NAD(P)H oxidse in the development of hhcys-induced glomerulr injury. Uninephrectomized Sprgue Dwley rts were fed folte-free diet for 8 weeks to produce hhcys nd the de novo cermide synthesis inhiitor myriocin or the NAD(P)H oxidse inhiitor pocynin ws dministrted. Rts with folte-free diet significntly incresed plsm Hcys levels, renl cermide levels, nd NAD(P)H oxidse ctivity ccompnied y mrked glomerulr injury. Tretment of rts with myriocin significntly reduced cermide levels nd improved glomerulr injury, s shown y decresed urinry lumin excretion nd reduced glomerulr dmge index. ECM components chnged towrds to norml levels with decresed tissue inhiitor of metlloproteinse-1 nd incresed mtrix metlloproteinse-1 ctivity. NAD(P)H oxidse ctivity nd Rc GTPse ctivity were reduced y 69 nd 66%, respectively. In rts treted with pocynin, similr eneficil effects in protecting glomeruli from hhcys-induced injury were oserved. These results support the view tht de novo cermide production is involved in Hcys-induced NAD(P)H oxidse ctivity in the kidney of hhcys rts nd indicte the importnt role of cermide-medited redox signling in hhcys-induced glomerulr injury in rts. Kidney Interntionl (6) 7, doi:1.138/sj.ki.51517; pulished online 1 My 6 KEYWORDS: homocysteine; sphingolipid; oxidtive stress; kidney; end-stge renl disese Correspondence: P-L Li, Deprtment of Phrmcology nd Toxicology, Medicl College of Virgini Medicl College of Virgini Cmpus, Virgini Commonwelth University, 41 N. 12th Street, Richmond, Virgini 23298, USA. E-mil: pli@mil1.vcu.edu Received 2 Septemer 5; revised 6 Ferury 6; ccepted 3 Mrch 6; pulished online 1 My 6 Hyperhomocysteinemi (hhcys) is emerging s criticl pthogenic fctor in the progression of end-stge renl disese nd in the development of crdiovsculr complictions relted to end-stge renl disese. 1 6 We hve demonstrted tht chronic elevtions of plsm Hcys levels even for just 2 weeks induced proteinuri, mesngil (MG) expnsion nd glomerulosclerosis, without elevtion of rteril lood pressure in uninephrotemized rts, indicting tht incresed plsm Hcys levels importntly contriute to the development of glomerulr disese independent of hypertension. 7 Despite sustntil evidence indicting the ssocition of hhcys nd end-stge renl disese, the mechnisms y which Hcys promotes the development of glomerulosclerosis remins unknown. Given the similrity of pthologicl chnges etween Hcys-induced glomerulr injury nd Hcys-induced rteril dmges such s endothelil injury, cell prolifertion, incresed mtrix formtion nd ggregted protoglycn, 8 1 the increse in plsm Hcys my lso ply crucil role in inititing nd fcilitting glomerulr injury in hypertensive nd end-stge renl disese individuls. Moreover, impired renl function will led to further increse in plsm Hcys, which, in turn, exggertes the progression of glomerulr injury, resulting in vicious cycle nd, consequently, in glomerulosclerosis. These Hcys involved vriety of cellulr, iochemicl, nd moleculr events re importntly relted to locl oxidtive stress Therefore, the oxidtive stress-medited pthogenic mechnism my contriute to Hcys-induced glomerulr injury. Recently, we hve demonstrted tht Hcys cutely increses superoxide (O 2 ) production vi NAD(P)H oxidse nd therey stimultes the formtion of tissue inhiitor of metlloproteinse-1 (TIMP-1) in rt MG cells, ultimtely resulting in the deposition of collgen. 14 We further demonstrted tht Hcys incresed de novo cermide synthesis in rt MG cells. This incresed cermide ctivted Rc GTPse nd enhnced NAD(P)H oxidse ctivity. 15 In previous studies, sphingolipids, in prticulr, cermide, ply very importnt role in cell memrne formtion, signl trnsduction, nd plsm lipoprotein metolisms, ll of 88 Kidney Interntionl (6) 7, 88 96

2 which hve n impct on the development of therosclerosis In ddition, cermide is considered s criticl signling molecule mediting the ctivtion of NAD(P)H oxidse in different cells. 15,19 Therefore, we hypothesized tht cermide-induced NAD(P)H oxidse ctivity my importntly prticipte in the development of hhcys-induced glomerulr injury. In the present study, we utilized myriocin to inhiit serine plmitoyl-coenzyme A trnsferse, the key enzyme for de novo cermide synthesis, nd exmined the role of endogenous-produced cermide in the development of Hcys-induced glomerulosclerosis in n experimentl hhcys niml model nd explored relted mechnisms. We lso treted hhcys rts with n NAD(P)H oxidse inhiitor, pocynin, to oserve its eneficil ctions on hhcys-induced glomerulr injury. Our results provided direct evidence, indicting tht incresed plsm Hcys stimultes cermide production nd NAD(P)H oxidse ctivity in the kidney of hhcys rts. We further demonstrted tht cermide-ctivted NAD(P)H oxidse promotes the formtion of TIMP-1 nd decrese mtrix metlloproteinse-1 (MMP-1) ctivity, ultimtely leding to glomerulosclerosis in hhcys rts. RESULTS Plsm totl Hcys levels significntly incresed in rts with folte-free diet High-pressure liquid chromtogrphy (HPLC) chromtogrms of ABD-F-derivtized stndrds of severl thiol compounds re presented in Figure 1. Stndrd thiol solution contins cysteine, Hcys, glutthione, cysteinylglycine, nd the internl stndrd TGA. Hcys hd retention time of 7.4 min, clerly seprted from other thiol compounds. In uninephrectomized Sprgue Dwley rts, n 8 week-folte-free diet significntly incresed plsm totl Hcys levels. Neither pocynin nor myriocin hd effect on the increse of Hcys levels induced y folte-free diet. The results were summrized in Figure 1. Additionlly, two groups of rts were continuously dministrted myriocin or pocynin with norml diet hd no significnt chnge in Hcys levels, suggesting tht either pocynin or myriocin only does not lter Hcys metolism. Effect of myriocin nd pocynin on glomerulr dmge induced y hhcys To determine whether the cermide redox pthwy contriutes to Hcys-induced glomerulr injury, we exmined urinry protein, lumin excretion, nd glomerulr morphology in rts fed folte-free diet. In prllel to increse in plsm totl Hcys, oth urinry protein nd urinry lumin were significntly incresed in rts fed folte-free diet with more drmtic increse in urinry protein (Figure 2). Morphologicl nlysis showed tht glomerulr extrcellulr mtrix (ECM) mrkedly incresed nd the glomerulr mesngium ws expnded with hypercellulrity, cpillry collpse, nd firous deposition in the glomerulus in hhcys rts (Figure 3). The semiquntittive injury score of glomeruli with the verge glomerulr dmge index ws LU LU LU Cys-gly Cys Hcys TGA sustntilly higher in rts fed folte-free diet thn in rts fed norml diet (Figure 3). However, tretment of hhcys rts with myriocin or pocynin mrkedly reduced urinry protein excretion, lumin excretion, nd glomerulr dmge index increse induced y hhcys. Myriocin or pocynin lone hd no effects on the urinry protein nd lumin excretion in rts with Glu STD Min Cys 35 3 Control group Cys-gly Hcys Glu TGA Cys Min 35 3 FF diet group Cys-gly 5 Hcys Glu TGA Min Plsm Hcys level (μm) Control Apo Myr FF FF+Apo FF+Myr Figure 1 Fluorescent HPLC chromtogrms of Hcys nd other thiols from stndrd solutions nd plsm. () HPLC chromtogrms showing stndrds (STD) contining 4 mm cysteine (Cys), mm Hcys nd glutthione (GSH), 5 mm cysteinylglycin (Cysgly), nd internl stndrd, 5 mm TGA nd Hcys, nd other thiols in the plsm from Sprgue Dwley (SD) rt with norml diet or folte-free diet (FF). () Averge plsm totl homocysteine concentrtions in different groups of rts on norml diet with or without myriocin (Myr) nd pocynin (Apo) tretments or folte-free diet with or without Myr nd Apo tretments (n ¼ 7). Po.5 compred with control. Kidney Interntionl (6) 7,

3 Urinry protein (mg/24 h) Control FF Control Apo Myr FF FF+Apo FF+Myr FF+Apo FF+Myr Urinry lumin (mg/24 h) Control Apo Myr FF FF+Apo FF+Myr Figure 2 Mesurement of urinry protein nd urinry lumin excretion. () Urinry protein nd () urinry lumin excretion in six different groups of rts fed norml diet or folte-free diet with or without myriocin nd pocynin tretments (n ¼ 7). Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. regulr chow. Bsed on these results, we conclude tht inhiition of de novo cermide synthesis or lockde of NAD(P)H oxidse does not hve effects on glomerulr structure nd function in rts on norml diet. In the following mechnism studies, therefore, only four groups of experiments re presented tht include norml diet or foltefree diet with or without myriocin or pocynin. Effects of myriocin nd pocynin on Hcys-incresed expression of TIMP-1 mrna nd protein levels By rel-time reverse trnscription-polymerse chin rection nd Western lot nlysis, TIMP-1 mrna nd protein levels in the renl cortex from rts with hhcys were incresed y 61. nd 47.5%, respectively (Figure 4 nd ). Both myriocin nd pocynin ttenuted Hcys-induced increse. The results were summrized in Figure 4c. Restortion of Hcys-reduced MMP-1 ctivity y myriocin nd pocynin tretments To exmine whether the regultion of MMP-1 ctivity, n importnt form of MMPs in the kidney, is ssocited with cermide redox signling pthwy in hhcys rts, MMP-1 ctivity ws mesured nd the results re summrized in Figure 5. MMP-1 ctivity in the renl cortex of hhcys rts ws mrkedly reduced to 54% of control group, nd myriocin nd pocynin tretments cn prtilly restore Hcys-reduced MMP-1 ctivity to 85 nd 81%, respectively. Score of glomerulr dmge index Apo Control Apo Myr Quntittion of cermide levels in the renl cortex of hhcys rts Using liquid chromtogrphy/mss spectrometry nlysis, we identified nd quntified six different cermides (C14, C16, C18, C, C22, C24). In these renl corticl smples, C16 nd C24 cermides re the most undnt species (98% of totl cermides). As summrized in Figure 6, in hhcys rts renl corticl cermide levels significntly incresed y 58%. When these rts were treted with myriocin, increses in renl corticl cermide levels were ttenuted y 98%. Moreover, there ws no significnt decrese in cermide levels in the pocynin-treted hhcys rts. On regulr chow, rts receiving pocynin hd no chnges in cermide levels in their kidneys, ut rts receiving myriocin exhiited decresed cermide level of 22% in their kidneys s compred with control rts (Figure 6). Myr FF FF+Apo FF+Myr Figure 3 Morphologicl fetures of the glomeruli from different groups of rts including norml diet or folte-free diet with or without myriocin nd pocynin tretments (n ¼ 7). () Photomicrogrphs (originl mgnifiction, 25) showing typicl glomerulr structure in control nd different tretment groups of rts. () Semiquntittive score of glomerulr dmge index (n ¼ 7). Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. 9 Kidney Interntionl (6) 7, 88 96

4 45 kd TIMP-1 mrna expression level kd- -TIMP-1 - -β-actin- Cermide level chnge (fold chnge of control) Control Apo Myr FF FF+Apo FF+Myr Figure 6 Effects of de novo cermide synthesis inhiitor myriocin nd NAD(P)H oxidse inhiitor pocynin on cermide production in the renl cortex of hhcys rts (n ¼ 6). Vlues re men7s.e. from seven smples ech group. Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. c TIMP-1 normlized to control Figure 4 TIMP-1 mrna nd protein expression levels in the renl cortex from different group of rts on norml diet, folte-free diet with or without myriocin nd pocynin tretment (n ¼ 7). () Summrized dt showing TIMP-1 mrna levels in the renl cortex from these rts. () Immunolot for TIMP-1 in the renl cortex from these rts. (c) Summrized dt showing chnges in TIMP-1 protein levels in the renl cortex from these groups of rts. Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. Eth-DNA fluorescence increse ( % vs control) Figure 7 NAD(P)H oxidse ctivity in the renl cortex from different groups of rts on norml diet or folte-free diet with or without myriocin nd pocynin tretments (n ¼ 7). Summrized dt showing the effect of Hcys nd inhiitors on NAD(P)Hdependent ctivity in these renl cortex. Dt were presented s percent increses in ethidium fluorescence compred to control. Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. MMP-1 ctivity chnge (% vs control) Figure 5 Effects of de novo cermide synthesis inhiitor myriocin nd NAD(P)H oxidse inhiitor pocynin on the MMP-1 ctivity in the renl cortex from these hhcys rts (n ¼ 7). Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. Blockde of enhnced NAD(P)H oxidse ctivity in hhcys rts y inhiition of de novo cermide synthesis The ctivity of NAD(P)H oxidse ws determined y fluorescence spectrometric ssy. Summrized dt showed tht NAD(P)H oxidse ctivity incresed y 61% in the renl cortex of rts fed folte-free diet compred with control rts. Administrtion of pocynin nd myriocin mrkedly ttenuted hhcys-induced NAD(P)H oxidse ctivity y 69 nd 55% (Figure 7). Expression of NAD(P)H oxidse suunits in the renl cortex To explore the mechnism y which myriocin ttenute Hcys-induced NAD(P)H oxidse ctivity in hhcys rts, we exmined expression levels of NAD(P)H oxidse suunits in the renl cortex of these treted rts. The representtive suunits p47 phox, p67 phox, non-phgocytic NAD(P)H oxidse (NOX)-2, nd NOX-4 of NAD(P)H oxidse were detected y Western lot nlysis. As shown in Figure 8, expression of these suunits in the renl cortex ws not significntly chnged. These results re summrized in Figure 8. Rc GTPse ctivtion is involved in cermide redox signling pthwy induced y hhcys As documented in Figure 9, hhcys produced significnt increse in GTP-ound Rc levels in the renl cortex of hhcys Kidney Interntionl (6) 7,

5 kd 21 kd - - Activted Rc 47- -p47 phox 21 kd - - Totl Rc NADPH oxidse suunits expression level (normlized to control) Control FF FF+Apo FF+Myr p47 phox phox p67 NOX-4 NOX-2 -p67 phox -NOX-4 -NOX-2 -β -Actin Figure 8 Expression of NAD(P)H oxidse suunits in the renl cortex from different groups of rts on norml diet or folte-free diet with or without myriocin nd pocynin tretment (n ¼ 7). () Immunolot for p47 phox, p67 phox, nd NOX-2 (gp91 phox ) nd NOX-4 in the homogentes from the renl cortex of these rts. () Summrized dt showing chnges in p47 phox, p67 phox, NOX-2 (gp91 phox ), nd NOX-4 in the renl cortex from different groups of rts. c Activted Rc Totl Rc normlized to control Figure 9 Rc GTPse ctivity in the renl cortex from different groups of rts fed on norml diet or folte-free diet with or without myriocin nd pocynin tretment (n ¼ 7). () Immunolot for Rc GTPse ctivity nd totl Rc GTPse expression levels. () Summrized dt showing chnges in Rc GTPse ctivity nd totl RC GTPse expression in renl cortex. Po.5 compred with control. Po.5 compred with the vlues otined from vehicle-treted hhcys rts. rts compred with the control group. However, dministrtion of myriocin significntly locked the Hcys-induced increse in ctivted Rc levels. The results re summrized in Figure 9. DISCUSSION Recently, we hve demonstrted tht chronic elevtions of plsm Hcys induces glomerulosclerosis nd indicted tht incresed Hcys levels ply n importnt role in the development of glomerulosclerosis. In in vitro experiments, Hcys ws found to increse O 2 production vi stimultion of de novo cermide synthesis nd susequent enhncement of Rc GTPse ctivity, which in turn stimultes the formtion of TIMP-1 in MG cells, consequently producing the deposition of collgen. 14,15 However, it remins unknown whether this cermide redox signling pthwy does contriute to the development of glomerulosclerosis ssocited with hhcys in vivo. In the present study, we performed series of experiments to investigte the role of this cermide redox signling pthwy in the hyperhomocysteinemic glomerulr injury in n experimentl hhcys niml model produced y feeding rts with folte-free diet. hhcys ssocited with folte-free diet induced significnt glomerulr injury It is well known tht Hcys is n intermedite metolite of methionine metolism nd is metolized y two pthwys: nmely, the re-methyltion pthwy tht regenertes methionine y cofctors folte nd vitmin B12, nd the trnssulfurtion pthwy tht degrdes Hcys into cysteine nd then turine y vitmin B6 nd other reltive coenzymes. Therefore, two mjor experimentl models to produce hhcys re widely used. 22 One is to use methionine-rich diet, wherey methionine cn e metolized to produce Hcys, nd nother is to use folte-free diet. Considering the tretment of hhcys in clinicl studies y dministrtion folic cid nd other vitmin supplementtion to reduce Hcys levels nd some concern out possile nonspecific effects of methionine in producing hhcys, the present study employed n niml model to induce hhcys y dietry folte restriction. Our results showed tht n 8-week folte-free diet significntly incresed plsm Hcys. In these rts, remrkle glomerulr dmge or sclerosis ws oserved, s shown y incresed injury score of glomeruli nd enhnced urinry protein nd lumin excretion. In these rts, urinry protein excretion incresed much higher thn lumin 92 Kidney Interntionl (6) 7, 88 96

6 excretion compred with those of control rts. It is possile tht in ddition to filtered lumin, some other proteins owing to injury of other tissues my enter into urine, suggesting tht renl tissue degenertion my occur in hhcys rts. It is well known tht glomerulr ccumultion of the ECM is the common pthologic feture of glomerulr injury, nd ltertions in the synthesis nd degrdtion of the ECM re importntly involved in the ccumultion of the ECM in the glomerulr. Recent studies hve demonstrted the pthologic role of mtrix-degrding MMPs nd their inhiitor TIMPs in glomerulr injury. 23 MMPs cn rek down collgens nd therey importntly regulte the mount of the ECM. 24,25 It hs een reported tht TIMP-1 plys key role in the regultion of the ECM ccumultion in glomerulr injury or sclerotic diseses. Excessive TIMP-1 cn mrkedly reduce the MMPs ctivity. 26,27 It is known tht MMP-1 is n importnt proteolytic enzyme tht degrdes collgens. 27,28 In the present study, it ws found tht oth TIMP-1 mrna expression nd protein levels in the renl cortex from hhcys rts were significntly incresed nd fluorescence resonnce energy trnsfer (FRET) ssy demonstrted tht the MMP-1 ctivity ws mrkedly reduced. These results indicted tht hhcys ssocited with folte-free diet induced n normlity of the ECM in the glomeruli, which my ultimtely led to severe significnt glomerulr injury. Role of de novo cermide synthesis in hhcys-induced glomerulr injury In hhcys rts fed folte-free diet, we tested whether cermide is involved in hhcys-induced NAD(P)H oxidse ctivity nd consequent glomerulr injury. Cermide, potent regultor of cell prolifertion, ctivtion, nd poptosis, plys n importnt role in lipoprotein ggregtion nd my promote fom cell formtion in therosclerosis. 29 It hs een implicted in the detrimentl ctions of mny different injury fctors in different cells nd tissues. 18,29,3 Recent reports hve shown tht serine plmitoyl-coenzyme A trnsferse inhiition y myriocin leds to reduction of plsm sphingomyelin, cermide production, nd therosclerosis in polipoprotein E-knockout mice. 31,32 More recently, we reported tht de novo cermide synthesis contriutes to Hcys-induced ctivtion of NAD(P)H oxidse ctivity in MG cells. 15 In this regrd, the possile pthologicl relevnce of cermide to glomerulr injury induced y Hcys ws proposed. The present study explored this possiility in experimentl hhcys rts. We utilized myriocin to inhiit the first step of de novo cermide synthesis nd to determine the impct of serine plmitoyl-coenzyme A trnsferse inhiition on lipid metolism nd glomerulosclerosis development in hhcys rts. It ws found tht renl corticl cermide levels in hhcys rts significntly incresed nd tht tretment of rts with myriocin mrkedly reduced cermide production in the kidneys of these rts. Menwhile, glomerulr injury in myriocin-treted rts ws mrkedly improved s shown y reduced urinry or luminuri nd decresed glomerulr dmge index. Correspondingly, ECM components chnged towrds to norml level with decresed TIMP-1 nd incresed MMP-1 ctivity. These results provide direct evidence indicting the importnt role of spingolipids, especilly cermide in hhcys-induced glomerulr injury in rts. It should e noted tht myriocin my hve nonspecific effects in the protection of glomeruli from injury ssocited with hhcys. However, severl lines of evidence support tht t the dose used with 8-week tretment myriocin my primrily work on de novo cermide synthesis. First, liquid chromtogrphy/mss spectrometry nlysis demonstrted tht myriocin significntly inhiited cermide production in rts with hhcys. Second, in the rts treted with myriocin, increse in NADPH oxidse ctivity y hhcys ws mrkedly locked. Bsed on our previous studies, 19 it is cermide, rther thn other sphingolipid tht stimultes NADPH oxidse. This further supports tht the ction of myriocin is through inhiition of cermide production. Third, recent studies hve indicted tht immunomodultion is not responsile for myriocin-induced inhiition of therogenesis in polipoprotein E-knockout mice. 32 In ddition, lthough immune cell dysfunction my ply some roles in the development of glomerulr sclerosis, it is well ccepted tht this immunopthogenic effect my not e mjor mechnism responsile for the development of glomerulr sclerosis. Incresed NAD(P)H oxidse ctivity contriutes to hhcys-induced glomerulr injury It is well known tht rective oxygen species decisively contriutes to cellulr signling, ffecting lmost ll spects of cellulr function including gene expression, prolifertion, migrtion, cell deth, nd sclerosis. 33,34 Although multiple enzymes contriute to oxidtive stress in different tissues or cells, recent studies hve een demonstrted tht NAD(P)H oxidse is mjor enzyme to produced O 2 in the kidney under physiologicl conditions. 35 NAD(P)H oxidse ws initilly chrcterized in neutrophils including memrne suunits p22 phox nd gp91 phox nd cytoplsmic suunits p47 phox, p4 phox, p67 phox, nd Rc GTPse. 36,37 In the pst few yers, fmily of gp91 phox -like proteins, termed the NOX proteins, hs lso een discovered which includes NOX1, NOX2 (gp91 phox ), NOX3, NOX4, nd NOX5. NOX4 is minly expressed in the kidney. 38 In recent studies, we hve demonstrted tht Hcys increses NAD(P)H oxidse-induced O 2 nd therey stimultes TIMP-1 in renl MG cells, ultimtely resulting in the deposition of collgen. The present study demonstrted in vivo tht NAD(P)H oxidse ctivity significntly incresed in the renl cortex of rts fed foltefree diet. When these hhcys rts were treted with n NAD(P)H oxidse inhiitor, pocynin, significnt eneficil effects to improve vrious hhcys-induced iochemicl or morphologicl lterntions in the glomeruli were oserved including decresed urinry protein excretion, improved glomerulr dmge, nd incresed MMP-1 ctivity. Interestingly, nlysis of NADPH oxidse ctivity showed tht Hcysinduces increse in NADPH ctivity ws lso mrkedly ttenuted in the presence of myriocin, de novo cermide Kidney Interntionl (6) 7,

7 synthesis inhiitor. These results provide direct evidence supporting the view tht O 2 production is ssocited with NADPH oxidse in hhcys rts. To explore the mechnisms y which Hcys ctivtes NAD(P)H oxidse in hhcys rts, we first exmined expression level of NAD(P)H oxidse suunits. Western lot nlysis showed tht the mount of NAD(P)H oxidse suunits in the renl cortex hd no chnge, no mtter whether the rts were treted y myriocin or pocynin, suggesting tht incresed NAD(P)H oxidse ctivity is not through incresed protein expression levels. Then, we tested whether Rc GTPse, criticl regultor, contriutes to incresed NAD(P)H oxidse ctivity in hhcys rts. It ws reported tht Rc-GTP intercts with the N-terminl of p67 phox for the ssemly of NAD(P)H oxidse suunits to ctivte this enzyme. 39,4 In our recent studies, cermide ws found to increse vi its de novo synthesis y Hcys tretment in rt MG cells. This incresed cermide-ctivted Rc GTPse nd enhnced NAD(P)H oxidse ctivity. The present study indicted tht Rc ctivity ws incresed y 88.3% in the kidney of hhcys rts. When myriocin ws dministrted, Hcys-induced increse in Rc GTPse ctivity ws mrkedly reduced, ut pocynin hd no effect. These results further support the view tht de novo cermide production is involved in Hcys-induced NAD(P)H oxidse ctivity y ctivtion of Rc GTPse in the kidney of hhcys rts. In summry, the present study demonstrtes tht cermide-ctivted NAD(P)H oxidse is responsile for glomerulr injury ssocited with hhcys in vivo nd tht ctivtion of NAD(P)H oxidse is ttriuted to incresed Rc-GTPse ctivity in the kidney of the hhcys rts. These results suggest new pthogenic pthwy contriuting to glomerulr injury ssocited with hhcys, which my direct towrd the development of new therpy of end-stge renl diseses relted to hhcys. MATERIALS AND METHODS Animls Experiments were performed using Sprgue Dwley rts ( g, 6 weeks old) purchsed from Hrln Inc. (Mdison, WI, USA). To speed up the dmging effect of Hcys on the glomeruli, the rts were uninephrectomized. After 1-week recovery period from uninephrectomy, the rts were mintined on regulr chow or foltefree diet, which ws purchsed from Dyets Inc. (Bethlehem, PA, USA) for 8 weeks. In dditionl groups, de novo cermide synthesis inhiitor, myriocin (.3 mg/kg/dy, intrperitonelly), or NAD(P)H oxidse inhiitor, pocynin (15 mg/kg/dy in drinking wter), ws continuously dministrted throughout the period feeding norml diet or folte-free diet. Myriocin nd pocynin doses were chosen ccording to recent reports 31,32 nd our previous dose-dependent experiments. All protocols were pproved y Institutionl Animl Cre nd Use Committee of oth Medicl College of Wisconsin nd Virgini Commonwelth University. During recording dys, lood nd 24-h urine smples were collected. Plsm totl Hcys ws mesured y fluorescence HPLC nlysis s descried. 41 Urinry protein concentrtion ws determined y BCA ssy kit (Bio-Rd, Hercules, CA, USA). Urinry lumin excretion ws mesured using rt lumin enzyme-linked immunosorent ssy quntittion kit (Bethyl Lortories, Montgomery, TX, USA). NAD(P)H oxidse ssy A dihydroethidium-sed fluorescence spectrometric ssy ws used to ssess O 2 production from NAD(P)H oxidse. 14,15 Dihydroethidium is oxidized specificlly y O 2 to yield ethidium, which inds DNA nd hs fluorescence t excittion/emission of 48/61 nm. Briefly, mg proteins were incuted with 1 mm dihydroethidium nd.5 mg/ml slmon test DNA (inds ethidium to mplify fluorescence signl) in ml phosphte-uffered sline. NADPH (1 mm) ws dded immeditely efore recording ethidium fluorescence y fluorescence microplte reder (FLX8, Bio-Tek, Winooski, VT, USA). The ethidium fluorescence increse ws used to represent NAD(P)H oxidse ctivity. Western lot nlysis Western lotting ws performed s descried. 14,15 Briefly, mg proteins were sujected to SDS-polycrylmide gel electrophoresis, trnsferred onto nitrocellulose memrne, nd locked. The memrne ws proed with primry monoclonl ntiodies: ntip47 phox, nti-p67 phox, nti-gp91 phox (1:1 dilution; BD Trnsduction Lortories, Lexington, KY, USA), nti-timp-1 (1:1 dilution; R&D systems, Minnepolis, MN, USA), or nti-nox-4 (1:5 dilution, Snt Cruz Biotech, Snt Cruz, CA, USA), overnight t 41C followed y incution with horserdish peroxidse-leled nti-mouse immunogloulin G or nti-got immunogloulin G. The immunorective nds were detected y chemiluminescence methods nd visulized on Kodk Omt film. Rc GTPse ctivtion ssy A specific pull-down experiment ws performed to determine Rc GTPse ctivtion using n Rc ctivtion ssy kit (Upstte, Lke Plcid, NY, USA) s descried. 15 Briefly, the renl cortex tissue ws lysed in Mg 2 þ lysis uffer. After preclened y glutthione grose, the pull-down proteins ( mg/smple) were incuted with 1 mg of PAK-1 PBD (glutthione S-trnsferse fusion protein, corresponding to p21-inding domin (PBD, residues of 67 15) of humn PAK-1) grose for one hour t 41C to ind Rc-GTP. Then, the pelleted eds were wshed three times with Mg 2 þ lysis uffer, resuspended in 25 ml of 2 Lemmli reducing smple uffer, nd oiled for 5 min. The smple mixtures were then loded onto SDSpolycrylmide gel electrophoresis gels. The ound ctive GTP-Rc ws nlyzed y Western lotting using nti-rc monoclonl ntiody. RNA extrction nd rel-time reverse trnscriptionpolymerse chin rection of TIMP-1 Totl RNA ws isolted from renl cortex using TRIzol regent (GIBCO, Life Technologies, Crlsd, CA, USA) ccording to the protocol descried y the mnufcturer. The mrna levels for TIMP-1 were nlyzed y rel-time quntittive reverse trnscription-polymerse chin rection using Bio-Rd icycler system (Bio- Rd, Hercules, CA, USA) ccording to the protocol descried y the mnufcturer. The mrna level of TIMP-1 ws normlized to the 18S mrna. The specific primers for TIMP-1 sed on the core sequence of rt TIMP-1 cdna (Accession numer NM-3254) were 5 -CCA GAA ATC ATC GAG ACC AC-3 (forwrd) nd 5 -CGG AAA CCT GTG GCA TTT-3 (reverse). 94 Kidney Interntionl (6) 7, 88 96

8 FRET ssy for MMP-1 ctivity mesurement MMP-1 ctivity ws mesured using EnzoLyte TM 5 MMP-1 ssy kit from AnSpec Inc. (Sn Jose, CA, USA). This kit uses 5-FAM (fluorophore)- nd QXL5 TM (quencher)-leled FRET peptide sustrte for continuous mesurement of the enzyme ctivities. In n intct FRET peptide, the fluorescence of 5-FAM is quenched y QXL5 TM. Upon the clevge of the FRET peptide y MMP-1, the fluorescence of 5-FAM is recovered, nd cn e continuously monitored t excittion/emission (49/5 nm). MMP-1 ctivity ws mesured ccording to the protocol descried y the mnufcturer. The chnge of enzyme ctivity of MMP-1 ws presented s percent chnge in reltive fluorescence unit vs control. Liquid chromtogrphy electrospry ioniztion tndem - mss spectrometry for quntittion of cermide Seprtion, identifiction nd quntittion of cermide in renl cortex were performed y liquid chromtogrphy/mss spectrometry. The HPLC is from HP 11 series equipped with inry pump, vcuum degsser, thermostted column comprtment, nd n utosmpler (Agilent Technologies, Wldronn, Germny). The HPLC seprtions were performed t 71C on n RP C18 Nucleosil AB column (5 mm, 7 mm 2 mm i.d.) from Mcherey Ngel (Düren, Germny). The moile phse ws grdient mixture formed s descried. 42 The tissue lipids were extrcted ccording to previous studies. 15,42 To void ny loss of lipids, the whole procedure ws performed in siliconized glsswre. Mss spectrometry detection ws crried out using n Ultim triple qudrupole instrument (Micromss, Mnchester, UK) operting under Mss- Lynx 3.5 nd configured with Z-spry electrospry ioniztion source. Source conditions were s descried previously. 42 Morphologicl exmintion The fixed kidneys were prffin-emedded, nd sections were prepred nd stined with periodic cid-schiff stin. 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