Báo cáo khoa học: "Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia" docx

J O U R N A L O F V eterin ary S cien ce J. Vet. Sci . (2002), 3(2), 115-121 ABSTRA CT 9) Prior ischem ia leads to resistan ce ag ainst su b sequ en t isch e m ic in su lts . Th e m e ch a n ism s th at und e rlie th is ad a p tive re sp o n se rem ain u n ide n tified. Th u s, w e stu d ied w h eth er th e re du c ed su sc ep tibility of m ice pre vio u sly s ubjec ted to th e isch e m ia to isc h em ia/ reperfu sion in jury is related w ith altered in fla m m ato ry respon ses. Th irty m in utes of bilateral kidney is ch em ia re su lts in sig n ifica n tly in crea se d pla sm a cre a tin in e an d bloo d u rea n itrogen le v els in B ALB /c m a le m ice. Th e re is severe d isru p tio n of actin cy to skele to n of pro x im a l tu bu la r cells in th e ou te r stripe of th e oute r m edu lla 24 h ou rs p o st-is chem ia . Wh e n m ice a re su bje cted to 30 m in u tes o f bila tera l isch e m ia 8 d ays la te r, th ere is n o in crea se in p la sm a cre a tin ine an d blo od ure a n itro g en lev els a n d th e post-is ch em ic dis ru p tio n of actin cy to skele to n o f p ro x im al tu b u lar ce lls is m u ch le ss. Infla m m a tory res po n se s h ave h igh ly im p lica ted w ith isc h em ia/rep erfu sio n in ju ry. Isc h em ia re su lts in th e in c rease d tiss u e m y elope r- ox ida se (MP O) a ctivity th at is a m ark er of leu k ocy te in filtra tion. Th e re is, h o w e v er, n o th e p ost-isch e m ic increase of MPO activity in kidneys previously su bje cte d to isch e m ia . P o st-isc h em ic expre ssio n o f tissu e in terce llu lar a dh e sio n m o lecu le -1 (ICAM-1) is gre a ter in th e kid n ey pre vio u sly sha m -o p era ted th a n in th e kidne ys previously subjected to ischem ia. In c on clu sio n , prior ischemia protects kidney function and m o rph o lo g y against subsequent ischemia 8 days later. The re sista n ce is a ssociated w ith th e reduced post-isch emic leu k o cy te in filtra tion d u e to th e re du c ed p o st-ische m ic ICAM-1 ex pre ssio n . Key w ord s : Ischemia , Inflamma tion, In tercellula r a dh esion m olecu le-1, K idn ey, M yelop eroxida se ＊ Corresponding author: Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Kwangju 500-757, Korea. Tel: 82-62-530-2831, Fax: 82-62-530-2809 E-mail: hjhan@chonnam.ac.kr Introduction Prior ischemic insult renders organs resistant to subsequent ischemia (1-5). We have previously reported that prior ischemia or ureteral obstruction prevents kidney against subsequent ischemia in the mouse kidney (6, 7). The mechanisms that underlie this adaptive response remain unidentified. Neutrophils are recruited to the sites of inflammation and play important roles in defense against infectious microorganisms by releasing superoxide and related radicals and enzymes such as proteases (8, 9). However, the excessive presence of these cells often augments injury by damaging surrounding normal tissues. Infiltration of neutrophils has been correlated with ischemia/reperfusion inju r y of sever a l organ s (10-12). Reduction of ren a l blood flow is one of im port an t fa ctors in post-ischemic acu te r ena l fa ilu re (13). The r educt ion of ren a l blood flow is affected by resu lts of in fla m m a t or y r ea ct ion s su ch a s en d ot h elia l dysfu n ct ion , leukocyte a dhesion , an d leu kocyte-endot helia l adh esion (14, 15). Con sidera ble eviden ce su ggest s tha t inhibition of the inflammatory reaction reduce the per tu rbation of r en a l blood flow and t he t ubula r dysfu n ct ion in du ced by isch em ia /r eperfusion (16, 17). We pr eviou sly repor ted th a t prior tra nsien t uretera l obstru ct ion ren ders the kidn ey resista n t to isch em ia and th a t th e kidn ey h as r educed post-isch em ic leukocyte in filtration (6). P ost-isch em ic tissu es gener at e in fla m m a tor y m edia t ors a n d u pregula te leu kocyte- en dot helia l adhesion molecu les, su ch as ICAM -1 wh ich ca n at tra ct a n d/or a ct ivat e leu kocytes, potent ia te sm all vessel occlusion, and promote further production of inflammatory mediators. Thus, we studied whether the reduced susceptibility of mice previously subjected to the ischemia to ischemia/ reperfusion injury is related with altered inflammatory responses. Our findings reveal that the kidneys previously exposed to ischemia are much less susceptible to subsequent ischemia than the kidneys previously sham-operated. This reduced injury is correlated with less leukocyte infiltration and lower post-ischemic ICAM-1 expression. Methods Animal preparation All experiments were performed in male BALB/c mice Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia Kwon-Moo Park and Ho-Jae Han* Department of Veterinary Physiology, College of Veterinary Medicine, Biotechnology Research Institute, Chonnam National University, Kwangju 500-757, Korea Received Mar. 6, 2002 / Accepted May 29, 2002 116 Kwon-Moo Park and Ho-Jae Han (Charles River Laboratory) weighing 20-25g. Mice were allowed free access to water and standard mice chow. Blood was drawn and a baseline level of serum creatinine deter- mined. Animals were anesthetized with pentobarbital sodium (50 mg/kg, ip) and administered 1 ml of 0.9 % NaCl (37 ℃ ) on the day of surgery (day 0). Body temperature was maintained at 36-38 ℃ . Kidneys were exposed through flank incisions. Animals were divided into 4 groups (Table 1). On day 0, mice were subjected to 30 minutes of bilateral renal ischemia by clamping both renal pedicles with non- traumatic microaneurysm clamps (preconditioned; Roboz). Some animals underwent sham surgery (non-preconditioned). The incisions were temporarily closed during ischemia or sham surgery. After 30 minutes the clamps were removed and reperfusion of the kidneys was visually confirmed. Animals were exposed to 30 minutes of either bilateral ischemia or sham surgery on day 8. Kidneys were harvested at indicated times on Figures. Kidneys were snap frozen in liquid nitrogen to use Western analysis or MPO activity, or were rinsed in phosphate buffered saline (PBS), and fixed in 4% paraformaldehyde for histological analysis. Renal functional parameters Seventy microliters of blood were taken from the retroorbital vein plexus at the times indicated on the Figures. Plasma creatinine or blood urea nitrogen (BUN) concentration was measured using a Beckman Creatinine Analyzer II or a spectrophotometer, respectively. Immunocytochemistry Sections were prepared from kidney fixed with 4% paraformaldehyde and were stained with phalloidin, which stains the actin cytoskeleton, or ICAM-1 as previously described(16, 18). Fluorescein isothiocyanate-labeled phalloidin (Phalloidin; 1:100) was obtained from Sigma(st. Ldlis, MO). Kidneys were perfused via the left ventricle with 30 ml of PBS for 2 minutes at 37 ℃ and then PLP (4% paraformaldehyde-75 mM L-lysine-10 mM sodium periodate) fixative. Kidneys were excised and placed in PLP overnight at 4 ℃ . Kidneys were then washed and stored in PBS containing 0.02% sodium azide at 4 ℃ . Fixed tissue was washed with PBS three times for 5 minutes each, placed overnight in PBS containing 30% sucrose, embedded in oxytetracycline compound (Sakura FineTek, Torrance, CA), frozen in liquid nitrogen, and then cut into 5 μ m sections using a cryotome. Sections were mounted on Fisher Superfrost Plus (Fisher LA, USA) microscope slides, dried in air and stored at -20 ℃ . For staining with phalloidin which stains actin cytoskeleton, sections were incubated in blocking buffer containing FITC-labelled phalloidin for 20 minutes at room temperature, washed three times in PBS for 5 minutes each and mounted with a 1: 1 m ixtu re of Vect ash i eld (Vector Laboratories LA, USA) and 0.3 M Tris HCl, pH 8.9. To detect ICAM-1, sections were dried, incubated in PBS containing 0.1% SDS for 5 minutes, washed in PBS for 10 minutes, and incubated in blocking buffer (PBS containing 2% BSA) for 20 minutes at room temperature. Sections were then incubated with antibody to ICAM-1, diluted in blocking buffer in a humidified chamber for 1 hour at room temperature. Sections were washed with PBS twice for 5 minutes each, with PBS containing 1.9% NaCl (high salt PBS) for 5 minutes and with PBS for 5 minutes. For negative controls, primary antibody was replaced with blocking buffer. Secondary antibodies were diluted in blocking buffer and placed on sections for 1 hour at room temperature, then washed twice in high salt PBS, once in PBS and mounted as described above. Images were viewed on a Nikon FXA epifluoescence microscope and collected using a digital camera (Hamamatusa Digital Camera). Myeloperoxidase (MPO) activity MPO activity, an index of tissue leukocyte infiltration, was measured in 24 hours post-ischemic kidney as previously described(6). Activity was normalized to protein concentration. Western Blot Analysis. Immunoblot were performed as previously described(7). ICAM-1 antibody was obtained from M.A. Arauot (Massa- chusetts General Hospital). Statistical analysis All results were expressed as mean ± S.E.M. The difference between two mean values was analyzed by ANOVA. A p<0.05 was taken as statistically significant. Table 1. Animal groups and procedures Groups n Initial procedure (day 0) Second procedure (day 8) Ⅰ Ⅱ Ⅲ Ⅳ 7 7 7 7 Sham bilateral ischemia Sham bilateral ischemia Bilateral ischemia Bilateral ischemia Sham bilateral ischemia Bilateral ischemia Sham bilateral ischemia Bilateral ischemia On day 0, animals were subjected to either 30 minutes of bilateral renal ischemia (preconditioned) or sham surgery (non- preconditioned) on day 0. Eight days after the first surgery, the animals were exposed to either 30 minutes of bilateral ischemia or sham-operation. Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia 117 Each group consisted of 7 animals as indicated in the Table 1. Results Prior ischemia preserves renal function and morphology from subsequent ischemia/reperfusion insult Renal ischemia/reperfusion results in severe loss of renal function. There are no changes of plasma creatinine and BUN levels on animals 24 hours after sham operation (Fig. 1). Thirty minutes of bilateral ischemia significantly increases the levels of plasma creatinine and BUN in the Group II animals (which is non-preconditioned). In the Group IV (which is preconditioned), ischemia on day 8 does not increase the levels of plasma creatinine when compared with the baseline levels (Fig. 1A). Before the subsequent ischemia on day 8, creatinine levels were indistinguishable from those at baseline in the all groups. The patterns of change in BUN closely paralleled those in creatinine in all experimental groups (Fig. 1B). On day 5 after second ischemic insult, the survival rate is 80% and 100% in the Group II and the Group IV animals, respectively. Renal ischemia/reperfusion results in disruption of actin cytoskeleton, fragmentation of microvilli and loss of cell polarity (19-22). We evaluated the effect of prior ischemia on post-ischemic histological changes using immunocytochemistry techniques. Sections were stained for phalloidin to identify the actin cytoskeleton (Fig. 2). There is normal phalloidin staining in sham-operated animals (Fig. 2). Ischemia on day 8 in the kidney non-preconditioned results in very severe widespread loss of the brush border actin in the S3 proximal tubular cells in the outer stripe of outer medulla (Fig. 2). In the animals preconditioned, changes in the post-ischemic kidney cytoskeleton actin staining is much less when compared with changes in the non-preconditioned (Fig. 2). Prior ischemia reduces post-ischemic myeloperoxidase (MPO) activity To evaluate whether leukocyte infiltration is associated with the increased resistance on the animals preconditioned, extent of tissue leukocyte infiltration was determined by tissue MPO activity which is an index of leukocyte infiltration. Twenty-four hours after ischemia, there is a dramatic increase in MPO activity in the animals non-preconditioned. By contrast, the ischemic preconditioning prevents most of the post-ischemic increase in tissue MPO activity (Fig. 3). There are no changes of MPO activity in the kidneys sham-operated on day 8 (Fig. 3). Prior ischemia reduces post-ischemic expression of tissue intercellular adhesion molecule-1 (ICAM-1) Since ICAM-1 can attract and/or activate leukocytes, potentiate small vessel occlusion, and promote further production of inflammatory mediators (16, 23, 24), we evaluated tissue ICAM-1 expression using Western blot and immunocytochemical analysis. Ischemia results in an increased expression of tissue ICAM-1. Six hours after ischemia on day 8, post-ischemic tissue ICAM-1 expression is greater in the kidneys non-preconditioned than in the kidneys pre- conditioned (Fig. 4). Sham-operation does not increase the tissue expression of ICAM-1 (Fig. 4). When the post- ischemic expression of ICAM-1 was immunohistologically evaluated, the expression levels is less in the kidneys preconditioned than in the kidneys non-preconditioned (Fig. 5). After ischemia, ICAM-1 is expressed in the outer stripe of the outer medulla which is most susceptible region to ischemia/reperfusion insult (Fig. 5). The post-ischemic expression of ICAM-1 is greater in the outer medullar than in the cortex. The post-ischemic expression of ICAM-1 is positively correlated with MPO activity. The post-ischemic ICAM-1 expression and MPO activity negatively correlates with the ischemia/reperfusion-induced renal functional and morphological injury. Discussion Our studies demonstrate that prior ischemia renders kidney resistant to the remote subsequent ischemia/ reperfusion insult 8 days later. Ischemia results in the increased leukocyte infiltration and ICAM-1 expression. Prior treatment of ischemia mitigates the post-ischemic leukocyte infiltration and ICAM-1 expression. Protective effects of preconditioning are transient and initially last only for a short period of time, i.e. less than 2 hours. A so-called "second window of protection" has been observed in some species, occurring 24 hours after the preconditioning stimulus in neurons and cardiomyocytes (25-27). Recent, we reported in mouse kidneys that the resistance induced by prior ischemia or transient ureteral obstruction was seen up to 15 or 8 days after the initial insults, respectively (6-7). In those studies, we have found that the protection is associated with the reduced post-ischemic activation of stress-activated protein kinase (SAPK) 1/2 or p38 and the increased actin cytoskeleton stability due to the increased heat shock protein-25 expression (6, 7). Ischemia and reperfusion in the kidney is characterized by marked structural and functional disruption of the proximal tubular epithelial cells in the outer stripe of the outer medulla (21, 22). Ischemia-induced functional damage is directly linked with disruption of actin cytoskeleton, since actin filament support brush-border or membrane solute transporters (19, 21). In these studies, we observed less post-ischemic disruption of the actin cytoskeleton in the proximal tubular cells in the outer stripes of the outer medulla in the kidneys previously subjected to ischemia when compared with the kidneys previously sham-operated. The increased cytoskeleton stability might preserve the polarity of proximal epithelial cells from ischemia/reperfusion injury and then reduce the renal functional disorders 118 Kwon-Moo Park and Ho-Jae Han F ig. 2 . Im m u n ocytoch emica l a ssessm en t of a ct in cytoskeleton on kidney section s. An im a ls were subject ed t o either sh am opera tion (a a n d b) or 30 m in u tes of bila teral isch em ia (c a nd d) on day 0. E ight y da ys a fter t he first su rgery, som e a n im a ls were exposed to eit her sha m -opera tion (a a n d c) or 30 m in u tes of bila ter al isch em ia (b a nd d). Th e k idn eys w ere h a rvested 24 h ou r s after t he second sur gery. The k idn ey section s were sta ined wit h anti-ph a lloidin a nt ibodies as descr ibed in Meth ods. Section s w ere ta ken from ou ter m edu lla. Fig . 1 . E ffect of pr ior trea tm ent of isch em ia on th e levels of (a ) pla sm a creatinin e a n d (b) blood urea n it rogen (BUN ) a fter an isch em ia /r eperfu sion . Anim als wer e su bject ed t o either sha m -op er ation (S) or 30 m in ut es of bila tera l isch em ia (I) on day 0. E ight y da ys a fter first su rgery, a nim als were subject ed to eit h er sh a m -opera t ion or 30 m in u tes of bila tera l isch em ia . Pla sm a crea tin in e an d BU N levels were m easu red 24 hou rs after ischemia . Va lu es a re expr essed a s m ea n ± S.E .M . *, p < 0.05 versus I-I. Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia 119 indu ced by isch em ic in ju ry. Ischemia /reperfusion resu lts in cytokin e production wh ich , in turn, can enhance leukocyte-endothelial adhesion in tera ction s in th e sm a ll vessels of t he ou ter m edu lla wit h a ssociated platelet activation , leukocyte adhesion, leukocyte in filtr a tion, an d r esultan t obstru ction (17). Follow in g ren al isch em ia , neu tr oph ils accum ulat e in th e out er st ripe of ou ter m edu lla . Th e in filt rated n eutroph ils h ar m cell st ru ct ur es or cell fu nct ion (28), plu g th e ascen din g va sa r ecta in th e ou ter st ripe of the ou ter m ed ulla , and fu rt her im p air th e oxygen su pply to t h e proxim a l st ra ight tu bu le, t he m ajor sit e of inju ry in ischemic ren al failu re (28-32). Con siderable eviden ce su ggest s that t he in h ibition of infla m m a tor y reaction r edu ce isch em ic in ju ry (16, 17). An ti-n eu t roph il seru m tr eat m ent Fig . 4. Effect of prior treatm ent of isch em ia on post-isch em ic expression of tissue in tercellula r a dhesion m olecu le-1 (ICAM-1). On day 0, a n im a ls were su bjected to eit h er sh am su r ger y (S) or 30 m in u tes of bila tera l isch em ia (I). E ight da ys aft er the first isch emia, th e an im a ls w ere subjected to either sh am -operation or 30 minutes of bila tera l isch em ia . Six h ou r s a fter th e secon d su rger y, k idneys w er e ha rvested and ICAM-1 expression w as determ in ed w ith an ti-ICAM-1 antibod y on West er n blot a n a lysis. Fig . 3 . E ffect of prior treat m ent of isch em ia on post-isch em ic leukocyt e in filt ration . Anim als were su bjected to either sh am -operation (S) or 30 min u tes of bila ter al isch em ia (I) on da y 0. Eigh t d ays a fter th e fir st surgery, t he an im a ls w er e su bjected to either sh am -opera tion or 30 m in utes of bila teral isch em ia . Twen ty-fou r hou rs a fter t he secon d su rgery, on da y 9, kid neys were h a rvested a nd m yeloperoxida se (MP O) a ct ivity wa s determ in ed. M PO act ivit y w as n or m alized to protein con cen tration . Va lues presen ted a re exp ressed a s m ea n ± S.E .M in 6 a n im als. *, p< 0.01 vs I-I. F ig . 5 . P ost-isch em ic exp ression of in tercellu la r a dh esion m olecu le-1 (ICAM-1) on k idn ey sect ion s. O n da y 0, a n im a ls were su bject ed t o eith er sh am su rger y (A) or 30 m in ut es of bilatera l ischemia (B). Eight da ys a fter t h e fir st ischemia , th e a nim als were su bject ed t o 30 m in utes of bilater al isch em ia . E ight h ou r s after th e secon d su rgery, kid neys were fixed with 4 % P LP fixa tive a nd sect ion s were pr epa red for im m u n ocytoch em ica l sta in in g. Section s w ere sta in ed with a nt i-ICAM-1 antibody as descr ibed in Meth od s. Arrows in dica te ICAM-1 expression . 120 Kwon-Moo Park and Ho-Jae Han reduces neutrophil cou nt and the n eu trophil-depleted a n im a ls are protected against ischemic ren al failu re (16). Fu rtherm ore we pr eviou sly observed th at a nti-ICAM-1 a n tibody protected m ice aga in st isch em ia /r ep er fusion in su lt a nd th a t m ice depleted of ICAM-1 gen e a re less su sceptible to isch em ic ren al in ju ry (33). In t h e presen t stu dies, w e observe th e profou n d redu ction of post-ischem ic leu kocyte- in filt ration an d expression of ICAM-1 in k idn eys previou sly su bjected t o ischemia. In recent, we observed that prior ureteral obstru ction resu lt s in t h e reduced post-isch em ic leu kocyte in filtr a tion an d con gestion in th e ou t er m edu lla in the kid ney (6). In con clu sion , we have dem on strated t h at p rior isch em ia ren der s t he kidn ey resista n t to isch em ia . Th e redu ced susceptibility in the a nim als previously su bjected to isch em ia m igh t be m edia ted by t h e mitiga ted post-isch emic infla- m m ation s, lea din g to isch em ia /r eperfusion -in du ced kidn ey da m ages. Th ese fin din gs ha ve im port a nt im plica tion s for un dersta ndin g of th e pat hoph ysiology of isch em ia-in du ced inju ry a nd provide a n ew pa ra digm for th e design of th era pies for isch em ic diseases. 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USA, 1994, 91, 812-6. . infiltration and lower post -ischemic ICAM-1 expression. Methods Animal preparation All experiments were performed in male BALB/c mice Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia Kwon-Moo. p < 0.05 versus I-I. Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia 119 indu ced by isch em ic in ju ry. Ischemia /reperfusion resu lts in cytokin e production wh ich. Kellerman P. S., Clark R. A., Hoilien C. A., Linas S. L., Prior Ischemic Treatment Renders Kidney Resistant to Subsequent Ischemia 121 and Molitoris B. A. Role of microfilaments in maintenance of proximal