急性百草枯中毒大鼠肾组织中PPAR-γ与TGF-β_1的表达及5-氨基水杨酸的治疗干预研究
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摘要
目的：百草枯（Paraquat，PQ），商品名有克无踪、对草快等，自20世纪50年代末发现其良好的除草作用后，一直被广泛应用于农业生产中。随着PQ在我国的大量使用，PQ中毒的发生率也逐年增高。临床研究发现，PQ毒性作用极强，且尚未发现特效解毒药，使得PQ成为我国继有机磷类及拟除虫菊酯类之后导致农药中毒的第三大种类。目前PQ染毒导致肾脏损伤的机制尚未完全明确。本实验研究通过测定PQ染毒大鼠的血清超氧化物歧化酶（Superoxide Dismutase，SOD）活力以及丙二醛（MaleicDialdehyde，MDA）、谷胱甘肽（L-Glutathione，GSH）含量，并应用免疫组织化学染色方法观察过氧化物酶增殖体激活受体-γ（PeroxisomeProliferator Activated Receptor-gamma， PPAR-γ）与转化生长因子-β1（Transforming Growth Factor-β1，TGF-β1）在PQ染毒大鼠肾组织中的表达以及5-氨基水杨酸（5-Aminosalicy acid，5-ASA）对上述指标的干预影响，进一步探讨PQ中毒对肾脏损伤的机制以及5-ASA对百草枯染毒大鼠肾损伤的保护机制，初步探索较大剂量5-ASA对于正常肾组织有无影响。
方法：由河北医科大学实验动物中心提供的健康成年雄性Wistar大鼠80只，体重在200-230g之间。动物室温度控制在20-26℃之间，湿度控制在40-70%之间，动物饲料由实验动物中心提供，饮用水为纯净水与盐酸配制的放置24小时后的酸化水（pH2.5-3.0）。正式实验前适应性饲养3天，根据体重将大鼠随机分为4组：①空白对照组（A组）20只；②单纯染毒组（B组）20只；③染毒+治疗组（C组）20只；④单纯5-ASA对照组（D组）20只。实验当天早晨8点，B、C两组分别给予80mg/kg的PQ灌胃染毒，A、D两组分别给予相同体积的双蒸水胃内灌注。上午10点，C、D两组分别给予75mg/kg的5-ASA胃内灌注，A、B两组分别给予相同体积的双蒸水胃内灌注。实验后第1天上午10点，C、D两组分别给予75mg/kg的5-ASA胃内灌注，A、B两组分别给予相同体积的双蒸水胃内灌注，每日1次，最长用药时间为7天。A、B、C、D各组于实验后1d、3d、7d、14d分别取5只大鼠，10%水合氯醛0.35mg/kg腹腔注射全身麻醉，开腹后髂总静脉取血3-4ml，4000r/h高速离心10min，取上层血清测定SOD活力以及MDA、GSH含量，应用统计学方法进行组间比较；迅速留取右侧肾脏，取肾组织进行HE染色，镜下观察超微组织结构，并进行免疫组织化学染色，观察PPAR-γ和TGF-β1在大鼠肾组织中的表达情况。
结果：
1.实验大鼠临床表现：A组（空白对照组）及D组（单纯5-ASA对照组）大鼠未出现任何中毒表现。B组（单纯染毒组）大鼠在灌胃染毒后2小时之内即出现中毒表现，染毒后3日内症状最为明显。较短时间内即可观察到竖毛，易激惹，厌食水症状；之后可出现呼吸困难，尿量减少，体重减轻，部分大鼠有血尿、稀便，口鼻及眼周血性分泌物。C组（染毒+治疗组）大鼠经5-ASA治疗后，中毒表现较B组为减轻，呼吸困难明显改善，体重下降不明显，血尿、稀便及口鼻、眼周血性分泌物少见。
2.实验大鼠血清学指标检测：
①B组大鼠血清SOD活力在染毒后的1d为最低，至7d时间点仍明显低于A组（P＜0.01)，后逐渐升高，至14d后与A组相比不再具有统计学意义（P＞0.05）。C组5-ASA明显增加了大鼠血清SOD活力，在染毒后的1d、3d、7d时间点均较B组明显升高，具有统计学意义（P＜0.01）；仅1d、3d时间点较A组具有统计学意义（P＜0.05）。D组与A组比较，SOD活力在染毒后的所有时间点均不具有统计学意义（P＞0.05）。
②B组大鼠血清MDA含量在染毒后的1d为最高，至7d时间点仍明显高于A组（P＜0.01）,后逐渐下降，至14d后与A组相比不具有统计学意义（P＞0.05）。C组5-ASA明显降低了中毒大鼠血清MDA含量，在染毒后的1d、3d、7d时间点均较B组明显降低（P＜0.01），仅1d、3d时间点较A组具有统计学意义（P＜0.05）。D组与A组比较，MDA含量在染毒后的所有时间点均不具有统计学意义（P＞0.05）。
③B组大鼠血清GSH含量在染毒后的1d为最低，至3d时间点仍明显低于A组（P＜0.01），后逐渐升高，至7d后与A组相比不再具有统计学意义（P＞0.05）。C组5-ASA明显增加了大鼠血清GSH含量，在染毒后的1d、3d、7d时间点均较B组明显升高，具有统计学意义（P＜0.01）；所有时间点较A组均不具有统计学意义（P＞0.05）。D组与A组比较，GSH含量在染毒后的所有时间点均不具有统计学意义（P＞0.05）。
3.实验大鼠肾组织形态学变化：
①大体形态：A组大鼠肾脏色泽红润正常，被膜无肿胀；B组大鼠肾脏颜色晦暗，被膜肿胀；C组大鼠肾脏与B组相比颜色较红，被膜肿胀较轻；D组大鼠肾脏色泽红润正常，被膜无肿胀。
②H E染色：A组大鼠肾组织中肾小球、肾小管、肾间质等各部分均未见异常；B组大鼠肾组织改变以肾小管为主，随着时间的推移，逐渐可见上皮细胞水肿、空泡变性、渗出坏死、管腔内可见红色坏死颗粒；C组大鼠肾组织改变与B组相类似，但程度有所减轻，发生时间有所延后；D组大鼠肾组织中肾小球、肾小管及肾间质均未见异常。
4.实验大鼠肾组织PPAR-γ与TGF-β1免疫组织化学检测结果：
4.1PPAR-γ在大鼠肾组织中的分布与表达：
①A组大鼠肾组织中，偶见PPAR-γ表达于髓质肾小管、集合管上皮细胞核中。②B组大鼠肾组织中，可见PPAR-γ少量表达于肾小管上皮细胞核中，偶见表达于肾血管内皮细胞核中。与A组相比，自染毒后1d时间点，PPAR-γ的表达有所增高，1d、3d时间点即具有统计学意义（P＜0.05），7d及14d具有显著统计学意义（P＜0.01）。③C组大鼠肾组织中，PPAR-γ主要表达在肾小管、集合管上皮细胞核中，其次表达在肾小球系膜细胞、肾血管内皮细胞等细胞核中，还可观察到上述细胞的胞浆内有少量表达。应用5-ASA治疗后，自染毒后1d开始该组大鼠肾组织中PPAR-γ的表达就显著增高，至14d仍有明显表达，与A组及B组相比均具有明显统计学意义（P＜0.01）。④D组大鼠肾组织中，偶见PPAR-γ表达于肾小管上皮细胞核中。与A组相比不具有统计学意义（P＞0.05）。
4.2TGF-β1在大鼠肾组织中的分布与表达：
①A组大鼠肾组织中，仅在肾小管上皮细胞的胞浆内偶可见TGF-β1表达，而在肾小球及肾小管周围毛细血管等肾间质区未见明显TGF-β1表达。②B组大鼠肾组织中，TGF-β1主要表达在肾小管上皮细胞的胞浆内，自1d时间点开始就显著增高，至7d到达高峰，且至14d仍有明显表达，与A组比较具有明显统计学意义（P＜0.01）。③C组大鼠肾组织中，TGF-β1亦主要表达在肾小管上皮细胞的胞浆内。应用5-ASA后自1d至14d其表达较B组减弱，具有统计学意义（P＜0.05）；与A组相比，自1d至14dTGF-β1的表达明显增高，具有显著统计学意义（P＜0.01）。④D组大鼠肾组织中，TGF-β1在肾小管上皮细胞的胞浆内偶见表达，未在肾小球及肾小管周围毛细血管区等肾间质部位见其表达。
结论：
1.PQ灌胃染毒可造成肾脏损伤，主要表现为肾小管上皮细胞损伤，表明PQ灌胃染毒是建立急性肾损伤动物模型的可靠方法。以80mg/kg灌胃染毒，剂量合适。
2.PQ中毒大鼠血浆中SOD活力降低， MDA含量增加，GSH含量减少，提示中毒大鼠体内存在过氧化损伤以及氧化—抗氧化失衡，可能是PQ导致肾脏损伤的机制之一。
3.PQ中毒大鼠肾组织中的TGF-β1主要在肾小管上皮细胞的胞浆中表达，可能是造成肾小管损伤的原因之一。而PPAR-γ主要表达于肾小管、集合管上皮细胞的胞核中，说明其可能具有抗衡并修复前者对肾小管损害的作用。
4.通过5-ASA治疗之后，可明显增加PQ中毒大鼠血清的SOD活力，降低MDA含量，升高GSH含量，从而减低氧化应激对于肾组织的损伤程度。
5.5-ASA可能是PPAR-γ的配体，通过与体内PPAR-γ的结合并使之激活，直接保护受损肾组织细胞，间接抑制体内TGF-β1的表达，从而减轻PQ所造成的肾脏损伤。
6.本实验研究未发现较大剂量5-ASA对于正常机体肾组织有损伤作用。
Objective：Paraquat(PQ),also known as Gramoxone,Esgram,et,whichherbicidal effector had been found excellent since the ends of1950s,has beenwidely used in agricultural production.Along with the widespread used in ourcountry,the incidence of PQ poisioning is increasing year by year. Throughclinical research we have found that PQ has highly toxic,but we have notfound an effective antidote.It makes PQ the third large species of pesticidepoisoning after organophosphorus and pyrethroid.Up to now the extrctmechanism of kidney toxity of PQ has not been fully enunciated.Ourexperimental study through determining the activity of superoxidedismutase(SOD),the content of maleic dialdehyde(MDA) and L-glutathione(GSH),observing the expressions of peroxisome proliferator activatedreceptor-gamma(PPAR-γ) and transforming growth factor-β1(TGF-β1) in theinjury kidney of PQ poisoned rats by the method of immunohisto-chemistry,further explore the mechanism of kidney toxity of PQ and the assessof kidney protection of5-ASA.We also make preliminar exploration toobserve5-ASA whether the impact of normal kidney tissue.
Methods：Eighty healthy adult male Wista rats,which the weight of200-300g,were provided by Hebei Medical University Experimental AnimalCenter.The temperature in animal room was controlled between20-26℃andthe huminity was controlled between40-70%.The animal feeds were providedby Experimental Animal Center,drinking water was acidified water which wasplaced24hours mixtured with pure water and hydrochloric acid (pH2.5-3.0).The rats were divided into4groups randomly after3day，s adaptivefeeding:①B lack Control Group(Group A):twenty rats;②PonsionedGroup(Group B):twenty rats;③P Q+5-ASA Group(Group C):twenty rats;④5-ASAControl Group(Group D):twenty rats。Group B and Group C were treated intragastrically with PQ (50mg/kg) by8o'clock in the experimerntal daymorning.Group A and Group D were treated with the same dose of doubledistilled water as Group B and Group C. By10o'clock Group C and Group Dwere treated intragastrically with5-ASA (75mg/kg),Group A and Group Bwere treated with the same dose of double distilled water as Group B andGroup C. Four Groups were treated in the same way with5-ASA (75mg/kg) ordouble distilled water each day and the longest medicine time is7days.Fiverats in four groups were anaesthesiaed by10%chloral hydrate on1stday,3rdday,7thday,14thafter PQ treatment respectively,then taken blood samples fromcommon iliac vein to determine SOD activity and MDA,GSH concentrationafter high speed centrifugation.We picked the right kidney of the rats rapidlyafter laparotomy for HE staining to oberserve the pathological changes andused IH staining to detect the expression of PPAR-γ and TGF-β1of kidneytissue.
Results：
1. Clinical manifestations of rats in experiment:the rats in Group A andGroup D did not appear the performance of PQ poisoning.The appearanceoccurred in2hours after PQ poisoning in rats of Group B,the most seriouslysymptom occurred within3days.In a short period of time we observedhorripilation,irritation,anorexia,hydrophobic;then the rats occurred dyspnea,hypourocrinia,low weight.Some rats had hematuresis,stools-loosing,eyes andnose bleeding.Rats in Group C,as compared with those in Group B,demonstrated the intoxication manifestations were alleviated signicantly afterthe treatment of5-ASA.Their symptoms of dyspnea,low weight,heaturesis,stools-loosing,eyes and nose bleeding were obviously relieved.
2. Serum measurement of rats in experiment:
①I n Group B,thelevels of serum SOD activity were the lowest on the1stday,the levels were still significantly low than those in Group A till the7thday(P＜0.01).Then the levels were increased gradually and there were nostatistical differences between Group B and Group A after the14th day(P＞0.05).In Group C,5-ASA obviously increased the levels of serum SOD activity,and the levels were remarkably higher than those in Group B on the1st,3rd,7th day,the differences between them had statistically significant(P＜0.01);there were only on the1st and3rd day statistical differences betweenGroup C and Group A(P＜0.05).The levels of serum SOD activity betweenGroup D and Group A had no statistically significant on the1st,3rd,7th and14th day(P＞0.05).
②In Group B,the levels of serum MDA content were the highest on the1st day,the levels were still significantly high than those in Group A till the7thday(P＜0.01). Then the levels were decreased gradually and there were nostatistical differences between Group B and Group A after the14th day(P＞0.05). In Group C,5-ASA remarkably decreased the levels of serum MDAcontent,and the levels were obviously lower than those in Group B on the1st,3rd,7th day,the differences between them had statistically significant(P＜0.01);there were only on the1st and3rd day statistical differences betweenGroup C and Group A(P＜0.05). The levels of serum MDA content betweenGroup D and Group A had no statistically significant on the1st,3rd,7th and14th day(P＞0.05).
③In Group B,the levels of serum GSH content were the lowest on the1stday,the levels were still significantly low than those in Group A till the3thday(P＜0.01).Then the levels were increased gradually and there were nostatistical differences between Group B and Group A after the7th day(P＞0.05).In Group C,5-ASA obviously increased the levels of serum GSHcontent,and the levels were remarkably higher than those in Group B on the1st,3rd,7th day,the differences between them had statistically significant(P＜0.01);there were no statistical differences between Group C and Group A fromstart to finish in the experiment(P＞0.05).The levels of serum GSH contentbetween Group D and Group A had no statistically significant on the1st,3rd,7th and14th day(P＞0.05).
3. Kidney tissue morphological changes in experiment:
①G ross appearance:The kidneys of Group A were normal and rosy,thecapsule was not swollen; in Group B,the colour of the kidneys was dark,the capsule was swollen;compared with Group B,the kidneys of Group C werebrighter,the swollen of surface was reduced;there were no abnormal in GroupD.
②HE staining:All parts of the kidneys in Group A were normal;in GroupB,the injuries were almost concertrated on tubular,as time goes by,we had seencell edema, vacuolar degeneration, exudative necrosis and red necrosisparticles in the lumen of tubular;the changes in kidney of Group C weresimilar with Group B,but the injuries relieved and the the time of occurrencehad been postponed;there were no abnormal in all parts of the kidneys inGroup D.
4. The expression of PPAR-γ and TGF-β1by IH staining in kidney
4.1The distribution and expression of PPAR-α in kidney of rats:
①In Group A,there was very weak expression of PPAR-γ in the nucleusof medullary tubular, collecting duct.②In Group B,PPAR-γ was expressed inthe nucleus of tubular,it also expressed in the nucleus of vascular endothelialcells occationally.Compared with Group A,the expression was increased fromthe1st day,the differences had statistically significant(P＜0.05) in the1st dayand the3rd day,there were obviously statistical differences from the7th day tothe14th day.③In Group C, PPAR-γ was mainly expressed in the nucleus oftubular,collecting duct,it also expressed in the nucleus of glomerularmesangial cells and vascular endothelial cells.We had found that a smallamount of PPAR-γ was expressed in the cytoplasm of cells above-mentioned.Since the1st day, the expression of PPAR-γ in kidney of rats was significantlyincreased after the treatment of5-ASA even till the14th day. There wereobviously statistical differences between Group B and Group A(P＜0.01).④P PAR-γ was occasionally found in nucleus of tubular epithelial cells inGroup D,the differeces had no statistically significant(P＞0.05).
4.2The distribution and expression of TGF-β1in kidney of rats:
①I n Group A,there was very weak expression of TGF-β1in thecytoplasm of tubular epithelial cells,we had not seen it in glomerular or kidneyrenal interstitium.②In Group B, TGF-β1was mainly expressed in the cytoplasm of tubular epithelial cells. Since the1st day, the expression ofPPAR-γ in kidney of rats was significantly increased,which reach the peak onthe7th day,and there were still obviously expression in the cytoplasm after the14th day.Compared with Group A, the differences had statistically significant(P＜0.01).③I n Group C, TGF-β1also was expressed in the cytoplasm oftubular epithelial cells mainly. Since the1st day, the expression of TGF-β1wassignificantly decreased after the treatment of5-ASA even till the14th day.Thedifferences had statistically significant(P＜0.05).Compared with Group A,theexpression of TGF-β1significantly increased from start to finish, thedifferences had apparently statistically significant(P＜0.01).④There was veryweak expression of TGF-β1in the cytoplasm of tubular epithelial cells inGroup D and was no expression in other parts of kidney.
Conclusions：
1.PQ intraperitoneally poisoning may cause acute kidney injury,and theprincipal damage is concentrate on renal tubule epithelial cells.We also cometo the conclusion that PQ intraperitoneally poisoning is a reliable method ofmaking a model of acute renal injury.The dose of80mg/kg is appropriated.
2. The activated of SOD is decreased,the content of MDA is inceased andthe content of GSH is decreased in the serum of PQ-poisoned rats.It promptsthat oxidative insult was one of the mechanisms of PQ-induced renal injuru.
3. TGF-β1was mainly expressed in the cytoplasm of tubular epithelialcells and it may be one of the causes of kidney tubular injury. PPAR-γ wasmainly expressed in the nucleus of medullary tubular, collecting duct and itmay resistant and recover the damage of TGF-β1.
4. The activated of SOD is obviously decreased,the content of MDA isinceased and the content of GSH is decreased through the treatment of5-ASA.We may conclude that5-ASA can alleviat the kidney injury.
5.5-ASA is possibly the ligand of PPAR-γ,which combines with andactivites PPAR-γ to protect injured kidney.It can also indirectly inhibit theexpression of TGF-β1to alleviat the kidney injury.
6. Through the experiment we find that larger doses of5-ASA may not harm the normal kidney.
方法：由河北医科大学实验动物中心提供的健康成年雄性Wistar大鼠80只，体重在200-230g之间。动物室温度控制在20-26℃之间，湿度控制在40-70%之间，动物饲料由实验动物中心提供，饮用水为纯净水与盐酸配制的放置24小时后的酸化水（pH2.5-3.0）。正式实验前适应性饲养3天，根据体重将大鼠随机分为4组：①空白对照组（A组）20只；②单纯染毒组（B组）20只；③染毒+治疗组（C组）20只；④单纯5-ASA对照组（D组）20只。实验当天早晨8点，B、C两组分别给予80mg/kg的PQ灌胃染毒，A、D两组分别给予相同体积的双蒸水胃内灌注。上午10点，C、D两组分别给予75mg/kg的5-ASA胃内灌注，A、B两组分别给予相同体积的双蒸水胃内灌注。实验后第1天上午10点，C、D两组分别给予75mg/kg的5-ASA胃内灌注，A、B两组分别给予相同体积的双蒸水胃内灌注，每日1次，最长用药时间为7天。A、B、C、D各组于实验后1d、3d、7d、14d分别取5只大鼠，10%水合氯醛0.35mg/kg腹腔注射全身麻醉，开腹后髂总静脉取血3-4ml，4000r/h高速离心10min，取上层血清测定SOD活力以及MDA、GSH含量，应用统计学方法进行组间比较；迅速留取右侧肾脏，取肾组织进行HE染色，镜下观察超微组织结构，并进行免疫组织化学染色，观察PPAR-γ和TGF-β1在大鼠肾组织中的表达情况。
结果：
1.实验大鼠临床表现：A组（空白对照组）及D组（单纯5-ASA对照组）大鼠未出现任何中毒表现。B组（单纯染毒组）大鼠在灌胃染毒后2小时之内即出现中毒表现，染毒后3日内症状最为明显。较短时间内即可观察到竖毛，易激惹，厌食水症状；之后可出现呼吸困难，尿量减少，体重减轻，部分大鼠有血尿、稀便，口鼻及眼周血性分泌物。C组（染毒+治疗组）大鼠经5-ASA治疗后，中毒表现较B组为减轻，呼吸困难明显改善，体重下降不明显，血尿、稀便及口鼻、眼周血性分泌物少见。
2.实验大鼠血清学指标检测：
①B组大鼠血清SOD活力在染毒后的1d为最低，至7d时间点仍明显低于A组（P＜0.01)，后逐渐升高，至14d后与A组相比不再具有统计学意义（P＞0.05）。C组5-ASA明显增加了大鼠血清SOD活力，在染毒后的1d、3d、7d时间点均较B组明显升高，具有统计学意义（P＜0.01）；仅1d、3d时间点较A组具有统计学意义（P＜0.05）。D组与A组比较，SOD活力在染毒后的所有时间点均不具有统计学意义（P＞0.05）。
②B组大鼠血清MDA含量在染毒后的1d为最高，至7d时间点仍明显高于A组（P＜0.01）,后逐渐下降，至14d后与A组相比不具有统计学意义（P＞0.05）。C组5-ASA明显降低了中毒大鼠血清MDA含量，在染毒后的1d、3d、7d时间点均较B组明显降低（P＜0.01），仅1d、3d时间点较A组具有统计学意义（P＜0.05）。D组与A组比较，MDA含量在染毒后的所有时间点均不具有统计学意义（P＞0.05）。
③B组大鼠血清GSH含量在染毒后的1d为最低，至3d时间点仍明显低于A组（P＜0.01），后逐渐升高，至7d后与A组相比不再具有统计学意义（P＞0.05）。C组5-ASA明显增加了大鼠血清GSH含量，在染毒后的1d、3d、7d时间点均较B组明显升高，具有统计学意义（P＜0.01）；所有时间点较A组均不具有统计学意义（P＞0.05）。D组与A组比较，GSH含量在染毒后的所有时间点均不具有统计学意义（P＞0.05）。
3.实验大鼠肾组织形态学变化：
①大体形态：A组大鼠肾脏色泽红润正常，被膜无肿胀；B组大鼠肾脏颜色晦暗，被膜肿胀；C组大鼠肾脏与B组相比颜色较红，被膜肿胀较轻；D组大鼠肾脏色泽红润正常，被膜无肿胀。
②H E染色：A组大鼠肾组织中肾小球、肾小管、肾间质等各部分均未见异常；B组大鼠肾组织改变以肾小管为主，随着时间的推移，逐渐可见上皮细胞水肿、空泡变性、渗出坏死、管腔内可见红色坏死颗粒；C组大鼠肾组织改变与B组相类似，但程度有所减轻，发生时间有所延后；D组大鼠肾组织中肾小球、肾小管及肾间质均未见异常。
4.实验大鼠肾组织PPAR-γ与TGF-β1免疫组织化学检测结果：
4.1PPAR-γ在大鼠肾组织中的分布与表达：
①A组大鼠肾组织中，偶见PPAR-γ表达于髓质肾小管、集合管上皮细胞核中。②B组大鼠肾组织中，可见PPAR-γ少量表达于肾小管上皮细胞核中，偶见表达于肾血管内皮细胞核中。与A组相比，自染毒后1d时间点，PPAR-γ的表达有所增高，1d、3d时间点即具有统计学意义（P＜0.05），7d及14d具有显著统计学意义（P＜0.01）。③C组大鼠肾组织中，PPAR-γ主要表达在肾小管、集合管上皮细胞核中，其次表达在肾小球系膜细胞、肾血管内皮细胞等细胞核中，还可观察到上述细胞的胞浆内有少量表达。应用5-ASA治疗后，自染毒后1d开始该组大鼠肾组织中PPAR-γ的表达就显著增高，至14d仍有明显表达，与A组及B组相比均具有明显统计学意义（P＜0.01）。④D组大鼠肾组织中，偶见PPAR-γ表达于肾小管上皮细胞核中。与A组相比不具有统计学意义（P＞0.05）。
4.2TGF-β1在大鼠肾组织中的分布与表达：
①A组大鼠肾组织中，仅在肾小管上皮细胞的胞浆内偶可见TGF-β1表达，而在肾小球及肾小管周围毛细血管等肾间质区未见明显TGF-β1表达。②B组大鼠肾组织中，TGF-β1主要表达在肾小管上皮细胞的胞浆内，自1d时间点开始就显著增高，至7d到达高峰，且至14d仍有明显表达，与A组比较具有明显统计学意义（P＜0.01）。③C组大鼠肾组织中，TGF-β1亦主要表达在肾小管上皮细胞的胞浆内。应用5-ASA后自1d至14d其表达较B组减弱，具有统计学意义（P＜0.05）；与A组相比，自1d至14dTGF-β1的表达明显增高，具有显著统计学意义（P＜0.01）。④D组大鼠肾组织中，TGF-β1在肾小管上皮细胞的胞浆内偶见表达，未在肾小球及肾小管周围毛细血管区等肾间质部位见其表达。
结论：
1.PQ灌胃染毒可造成肾脏损伤，主要表现为肾小管上皮细胞损伤，表明PQ灌胃染毒是建立急性肾损伤动物模型的可靠方法。以80mg/kg灌胃染毒，剂量合适。
2.PQ中毒大鼠血浆中SOD活力降低， MDA含量增加，GSH含量减少，提示中毒大鼠体内存在过氧化损伤以及氧化—抗氧化失衡，可能是PQ导致肾脏损伤的机制之一。
3.PQ中毒大鼠肾组织中的TGF-β1主要在肾小管上皮细胞的胞浆中表达，可能是造成肾小管损伤的原因之一。而PPAR-γ主要表达于肾小管、集合管上皮细胞的胞核中，说明其可能具有抗衡并修复前者对肾小管损害的作用。
4.通过5-ASA治疗之后，可明显增加PQ中毒大鼠血清的SOD活力，降低MDA含量，升高GSH含量，从而减低氧化应激对于肾组织的损伤程度。
5.5-ASA可能是PPAR-γ的配体，通过与体内PPAR-γ的结合并使之激活，直接保护受损肾组织细胞，间接抑制体内TGF-β1的表达，从而减轻PQ所造成的肾脏损伤。
6.本实验研究未发现较大剂量5-ASA对于正常机体肾组织有损伤作用。
Objective：Paraquat(PQ),also known as Gramoxone,Esgram,et,whichherbicidal effector had been found excellent since the ends of1950s,has beenwidely used in agricultural production.Along with the widespread used in ourcountry,the incidence of PQ poisioning is increasing year by year. Throughclinical research we have found that PQ has highly toxic,but we have notfound an effective antidote.It makes PQ the third large species of pesticidepoisoning after organophosphorus and pyrethroid.Up to now the extrctmechanism of kidney toxity of PQ has not been fully enunciated.Ourexperimental study through determining the activity of superoxidedismutase(SOD),the content of maleic dialdehyde(MDA) and L-glutathione(GSH),observing the expressions of peroxisome proliferator activatedreceptor-gamma(PPAR-γ) and transforming growth factor-β1(TGF-β1) in theinjury kidney of PQ poisoned rats by the method of immunohisto-chemistry,further explore the mechanism of kidney toxity of PQ and the assessof kidney protection of5-ASA.We also make preliminar exploration toobserve5-ASA whether the impact of normal kidney tissue.
Methods：Eighty healthy adult male Wista rats,which the weight of200-300g,were provided by Hebei Medical University Experimental AnimalCenter.The temperature in animal room was controlled between20-26℃andthe huminity was controlled between40-70%.The animal feeds were providedby Experimental Animal Center,drinking water was acidified water which wasplaced24hours mixtured with pure water and hydrochloric acid (pH2.5-3.0).The rats were divided into4groups randomly after3day，s adaptivefeeding:①B lack Control Group(Group A):twenty rats;②PonsionedGroup(Group B):twenty rats;③P Q+5-ASA Group(Group C):twenty rats;④5-ASAControl Group(Group D):twenty rats。Group B and Group C were treated intragastrically with PQ (50mg/kg) by8o'clock in the experimerntal daymorning.Group A and Group D were treated with the same dose of doubledistilled water as Group B and Group C. By10o'clock Group C and Group Dwere treated intragastrically with5-ASA (75mg/kg),Group A and Group Bwere treated with the same dose of double distilled water as Group B andGroup C. Four Groups were treated in the same way with5-ASA (75mg/kg) ordouble distilled water each day and the longest medicine time is7days.Fiverats in four groups were anaesthesiaed by10%chloral hydrate on1stday,3rdday,7thday,14thafter PQ treatment respectively,then taken blood samples fromcommon iliac vein to determine SOD activity and MDA,GSH concentrationafter high speed centrifugation.We picked the right kidney of the rats rapidlyafter laparotomy for HE staining to oberserve the pathological changes andused IH staining to detect the expression of PPAR-γ and TGF-β1of kidneytissue.
Results：
1. Clinical manifestations of rats in experiment:the rats in Group A andGroup D did not appear the performance of PQ poisoning.The appearanceoccurred in2hours after PQ poisoning in rats of Group B,the most seriouslysymptom occurred within3days.In a short period of time we observedhorripilation,irritation,anorexia,hydrophobic;then the rats occurred dyspnea,hypourocrinia,low weight.Some rats had hematuresis,stools-loosing,eyes andnose bleeding.Rats in Group C,as compared with those in Group B,demonstrated the intoxication manifestations were alleviated signicantly afterthe treatment of5-ASA.Their symptoms of dyspnea,low weight,heaturesis,stools-loosing,eyes and nose bleeding were obviously relieved.
2. Serum measurement of rats in experiment:
①I n Group B,thelevels of serum SOD activity were the lowest on the1stday,the levels were still significantly low than those in Group A till the7thday(P＜0.01).Then the levels were increased gradually and there were nostatistical differences between Group B and Group A after the14th day(P＞0.05).In Group C,5-ASA obviously increased the levels of serum SOD activity,and the levels were remarkably higher than those in Group B on the1st,3rd,7th day,the differences between them had statistically significant(P＜0.01);there were only on the1st and3rd day statistical differences betweenGroup C and Group A(P＜0.05).The levels of serum SOD activity betweenGroup D and Group A had no statistically significant on the1st,3rd,7th and14th day(P＞0.05).
②In Group B,the levels of serum MDA content were the highest on the1st day,the levels were still significantly high than those in Group A till the7thday(P＜0.01). Then the levels were decreased gradually and there were nostatistical differences between Group B and Group A after the14th day(P＞0.05). In Group C,5-ASA remarkably decreased the levels of serum MDAcontent,and the levels were obviously lower than those in Group B on the1st,3rd,7th day,the differences between them had statistically significant(P＜0.01);there were only on the1st and3rd day statistical differences betweenGroup C and Group A(P＜0.05). The levels of serum MDA content betweenGroup D and Group A had no statistically significant on the1st,3rd,7th and14th day(P＞0.05).
③In Group B,the levels of serum GSH content were the lowest on the1stday,the levels were still significantly low than those in Group A till the3thday(P＜0.01).Then the levels were increased gradually and there were nostatistical differences between Group B and Group A after the7th day(P＞0.05).In Group C,5-ASA obviously increased the levels of serum GSHcontent,and the levels were remarkably higher than those in Group B on the1st,3rd,7th day,the differences between them had statistically significant(P＜0.01);there were no statistical differences between Group C and Group A fromstart to finish in the experiment(P＞0.05).The levels of serum GSH contentbetween Group D and Group A had no statistically significant on the1st,3rd,7th and14th day(P＞0.05).
3. Kidney tissue morphological changes in experiment:
①G ross appearance:The kidneys of Group A were normal and rosy,thecapsule was not swollen; in Group B,the colour of the kidneys was dark,the capsule was swollen;compared with Group B,the kidneys of Group C werebrighter,the swollen of surface was reduced;there were no abnormal in GroupD.
②HE staining:All parts of the kidneys in Group A were normal;in GroupB,the injuries were almost concertrated on tubular,as time goes by,we had seencell edema, vacuolar degeneration, exudative necrosis and red necrosisparticles in the lumen of tubular;the changes in kidney of Group C weresimilar with Group B,but the injuries relieved and the the time of occurrencehad been postponed;there were no abnormal in all parts of the kidneys inGroup D.
4. The expression of PPAR-γ and TGF-β1by IH staining in kidney
4.1The distribution and expression of PPAR-α in kidney of rats:
①In Group A,there was very weak expression of PPAR-γ in the nucleusof medullary tubular, collecting duct.②In Group B,PPAR-γ was expressed inthe nucleus of tubular,it also expressed in the nucleus of vascular endothelialcells occationally.Compared with Group A,the expression was increased fromthe1st day,the differences had statistically significant(P＜0.05) in the1st dayand the3rd day,there were obviously statistical differences from the7th day tothe14th day.③In Group C, PPAR-γ was mainly expressed in the nucleus oftubular,collecting duct,it also expressed in the nucleus of glomerularmesangial cells and vascular endothelial cells.We had found that a smallamount of PPAR-γ was expressed in the cytoplasm of cells above-mentioned.Since the1st day, the expression of PPAR-γ in kidney of rats was significantlyincreased after the treatment of5-ASA even till the14th day. There wereobviously statistical differences between Group B and Group A(P＜0.01).④P PAR-γ was occasionally found in nucleus of tubular epithelial cells inGroup D,the differeces had no statistically significant(P＞0.05).
4.2The distribution and expression of TGF-β1in kidney of rats:
①I n Group A,there was very weak expression of TGF-β1in thecytoplasm of tubular epithelial cells,we had not seen it in glomerular or kidneyrenal interstitium.②In Group B, TGF-β1was mainly expressed in the cytoplasm of tubular epithelial cells. Since the1st day, the expression ofPPAR-γ in kidney of rats was significantly increased,which reach the peak onthe7th day,and there were still obviously expression in the cytoplasm after the14th day.Compared with Group A, the differences had statistically significant(P＜0.01).③I n Group C, TGF-β1also was expressed in the cytoplasm oftubular epithelial cells mainly. Since the1st day, the expression of TGF-β1wassignificantly decreased after the treatment of5-ASA even till the14th day.Thedifferences had statistically significant(P＜0.05).Compared with Group A,theexpression of TGF-β1significantly increased from start to finish, thedifferences had apparently statistically significant(P＜0.01).④There was veryweak expression of TGF-β1in the cytoplasm of tubular epithelial cells inGroup D and was no expression in other parts of kidney.
Conclusions：
1.PQ intraperitoneally poisoning may cause acute kidney injury,and theprincipal damage is concentrate on renal tubule epithelial cells.We also cometo the conclusion that PQ intraperitoneally poisoning is a reliable method ofmaking a model of acute renal injury.The dose of80mg/kg is appropriated.
2. The activated of SOD is decreased,the content of MDA is inceased andthe content of GSH is decreased in the serum of PQ-poisoned rats.It promptsthat oxidative insult was one of the mechanisms of PQ-induced renal injuru.
3. TGF-β1was mainly expressed in the cytoplasm of tubular epithelialcells and it may be one of the causes of kidney tubular injury. PPAR-γ wasmainly expressed in the nucleus of medullary tubular, collecting duct and itmay resistant and recover the damage of TGF-β1.
4. The activated of SOD is obviously decreased,the content of MDA isinceased and the content of GSH is decreased through the treatment of5-ASA.We may conclude that5-ASA can alleviat the kidney injury.
5.5-ASA is possibly the ligand of PPAR-γ,which combines with andactivites PPAR-γ to protect injured kidney.It can also indirectly inhibit theexpression of TGF-β1to alleviat the kidney injury.
6. Through the experiment we find that larger doses of5-ASA may not harm the normal kidney.
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3Be1lomo R，Ronco C，Ke1hm JR，et a1.Acute rena1failure definition，outcome measures， animal model， fluid therapy and informationtechnology needs：the Second International Consensus Conference of theAcute Dialysis Quality Initiative (ADQI)Group [J].Crit Care，2004，8(4)：R204-212
4Chertow GM，Burdick E，Honour M，et al. Acute kidney injury，mortality，length of stay，and cost in hospitalized patients [J].J Am Soc Nephrol，2005，16(11)：3365-3370
5Perrone RD，Madias NE，Levey AS. Serum creatinine as an index of renalfunction：new insight into old concepts [J].Clin Chem，1992，38(10)：1933-1953
6Ronco C，Levin A，Warnock DG，et al. Improving outcomes from acutekidney injury(AKI)：report on an initiative[J].Int J Artif Organs，2007，30(5)：373-376
7Mehat RL，Kellum JA，Shah SV，et al. Acute Kidney Injury Network：report of an initiative to improve outcomes in acute kidney injury. CritCare，2007，11：R31
8陈香美.肾脏病学科专业新进展及展望(A).解放军医学杂志，2010，35（9）：1409-1052
9滕杰，丁小强.急性肾损伤的定义、诊断及治疗现状[A].诊断学理论与实践，2007，6（6）：485-488
10Srisawat N，Hoste EE，Kellum JA. Modern classification of acute kidneyinjury[J].Blood Purif，2010，29(3)：300-307
11易著文，刘琳.急性肾损伤的定义、诊断及治疗[A].临床儿科杂志，2009，27（4）：301-306
12Mehta RL，Kellum JA，Shah SV，et al. Acute renal failure-definition，outcome measures， animal moldels， fluid therapy and informationtechnology needs：the Second International Consensus Conference of theAcute Dialysis Quality Initiative(ADQI) Group [J].Crit Care Med，2007，
35(8)：1837-1843
13Waikar SS，Boncentre JV.Biomarkers for the diagnosis of acute kidneyinjury.Curr Opin Nephrol Hypertens，2007，16：557-564
14Vaidya VS，Ferguson MA，Bonventre JV. Biomarkers of acute kidneyinjury. Annu Rev Pharmacol Toxicol，2008，48：463-493
15Nguyen M T，Decarajan P. Biomarkers for the early detection of acutekidney injury[J].Pediatr Nephrol，2008，23：2151-2157
16Vaidya VS，Ramirez V，Ichimura T，ct al.Urinary kidney injurymolccule-1：a sensitive quantitative biomarker for early detection ofkidney tubular injury[J].Am J Physiol Renal Physiol，2006，290(2)：F517-F529
17Dagher PC，Herget-Rosenthal S，Ruehm SG，et al. Newly developedtechniques to study and diagnose acute renal failure[J].J Am SocNephrol，2003，14(8)：2188-2198
18Parikh CR，Abraham E，Ancukiewicz M，et al.Urine IL-18is an earlydiagnostic marker for acute kidney injury and predicts mortality in theintensive care unit[J].J Am Soc Nephrol，2005，16(10)：3046-3052
19Muramatsu Y，Tsujie M，Kohda Y，et al. Early detection of cysteine richprotein61(CYR61，CCNI)in urine following renal ischemic reperfusioninjury [J].Kidney Int，2002，62(5)：1601-1610
20Du Cheyron D，Daubin C，Poggioli J，et al.Urinary measurement ofNa+/H+exchanger isoform3(NHE3) protein as new marker of tubuleinjury critically ill patients with ARF[J].Am J Kidney Dis，2003，42(3)：497-506
21金霞，王红云.胱抑素C在临床中的运用研究[B].中国健康月刊（学术版），1005-0510（2011）08-0343-021
22戎殳综述，梅长林审校.急性肾损伤定义诊断及治疗进展[A].Chinese Journal of Practical Internal Medicine Nov.2006Vol.26No.21，1005-2194（2006）21-1740-04
23蒋芬，梁馨苓.急性肾损伤临床研究进展[J].实用医学杂志2011年第
27卷第6期：933-935
24张祯，王晓明.血清胱抑素C量血蛋白检测对早期糖尿病损害的意义[J].中国医学检验杂志，2008，29（1）：3
25王鹏飞，沈玲红，何奔.急性肾损伤生物标志物的研究进展[C].J InternMed Concepts Pract2010，Vol.5，No.3：268-272
26Coca SG，Parikh CR.Urinary biomarkers for acute kidney injury:：perspectives on translation. Chin J A Soc Nephrol，2008，4：481-490
27Herget-Rosenthal S，Pietruck F，Volbracht L，et al. Serum cystatin C—asuperior marker of rapidly reduced glomerular filtration afteruninephrectomy in kidney donors compared to creatine [J].Clin Nephrol，2005，64（1）：41-46
28Ichimura TI，Hung CC，Yang SA，et al. Kidney injury molecule-1：atissue and urinary biomarker for nephrotoxicant-induced renal injury[J].Am J Physiol Renal Physiol，2006，286(3)：552-563
29Herget-Rosenthal S，Marggraf G，Husing J，et al. Early detection of acuterenal failure by serum cystatin C [J].Kidney Int，2004，66(3)：1115-1122
30吴亚君，施岚，范亚平，等.尿液西司他丁测定与肾小球损害的相关性研究[J].放射免疫学杂志，2004，17（3）：232-233
31Dharnidharka T，Kwon C，Stevens G，et al. Serum cystatin C is superiorto serum creatinine as a marker of kidney function：A meta-analysis[J].Am J Kidney Disease，2002，40(2)：221-226
32Michael Bennett，：Catherine L Dent，Qing Ma，et al. Urine NGALPredicts Severity of Acute Kidney Injury After Cardiac Surgery：AProspective Study [J]. Clin J Am Soc Nephrol，2008，3(3)：665-669.
33Han WK，Bailly V，Abichandani R，et al. Kidney injury molecule-1(KIM-1)：a novelbiomarker for human renal proximal tubule injury[J].Kidney Int2002，62(1)：237-244
34Zhang Z，Humphreys BD，Boruentre JV. Shedding of the urinarybiomarker kidney injury molecule-1(KIM-1) is regulated by MAPkinases and juxtamembrane region [J].J Am Soe Nephrol，2007，18(10)：2701-2711
35Liangos O，Perianayagam M C, Vaidya, et al.Urinary N-acetyl-beta(D)-glucosaminidase activity and kidney injury molecule-1level areassociated with adverse outcomes in acute renal failure [J].J Am SocNephrol，2007，18(3)：904-912
36Ostermann M，Chang RW.Acute kidney injury in the intensive care unitaccording to RIFLE[J].Crit Care Med，2007,35（8）：1837-1843
37Mishra J，Dent C，Tarabishi R，et al.Neutrophil gelatinase-associatedlipocalin（NGAL） as a biomarker for acute renal injury after cardiacsurgeru[J].Lancet，2005,365（9466）：1231-1238
38Parikh CR，Jani A，Melnikov VY，et al.Urinary interleukin18is a markerof human acute tubular necrosis[J].Am J Kidney Dis，2004,43（3）：405-414
39Washburn KK，Zappitelli M，Arikan AA，et al.Urinary interleukin-18isan acute kidney injury biomarker in crttically ill children[J].Nephrol DialTransplant，2008，23（2）：566-572
40Du Cheyron D，Daubin C，Poggioli J，et al.Urinary measurement of Na＋/H＋exchanger isoform3（NHE-3）protein as new marker of tubuleinjury in critically ill patients with ARF[J].Am J Kidney Dis，2003,42（3）：497-506
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42Vaikya VS，Ferguson MA，Bonventre JV.Biomarkers of acute kidneyinjury[J].Annu Rev Pharmacol Toxicol，2008,48:463-493
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1Wamoch DG. Towards a definition and classification of acute kidneyinjury[J].J Am Soc Nephrol，2005，16(11)：3149-3150
2Lassnigg A，Schmidlin D，Mouhieddine M，et al. Minimal-changes ofserum creatinine predict prognosis in patients after cardiothoracic surgery：a prospective cohort study[J].J Am Soc Nephol，2004，15(6)：1597-1605
3Be1lomo R，Ronco C，Ke1hm JR，et a1.Acute rena1failure definition，outcome measures， animal model， fluid therapy and informationtechnology needs：the Second International Consensus Conference of theAcute Dialysis Quality Initiative (ADQI)Group [J].Crit Care，2004，8(4)：R204-212
4Chertow GM，Burdick E，Honour M，et al. Acute kidney injury，mortality，length of stay，and cost in hospitalized patients [J].J Am Soc Nephrol，2005，16(11)：3365-3370
5Perrone RD，Madias NE，Levey AS. Serum creatinine as an index of renalfunction：new insight into old concepts [J].Clin Chem，1992，38(10)：1933-1953
6Ronco C，Levin A，Warnock DG，et al. Improving outcomes from acutekidney injury(AKI)：report on an initiative[J].Int J Artif Organs，2007，30(5)：373-376
7Mehat RL，Kellum JA，Shah SV，et al. Acute Kidney Injury Network：report of an initiative to improve outcomes in acute kidney injury. CritCare，2007，11：R31
8陈香美.肾脏病学科专业新进展及展望(A).解放军医学杂志，2010，35（9）：1409-1052
9滕杰，丁小强.急性肾损伤的定义、诊断及治疗现状[A].诊断学理论与实践，2007，6（6）：485-488
10Srisawat N，Hoste EE，Kellum JA. Modern classification of acute kidneyinjury[J].Blood Purif，2010，29(3)：300-307
11易著文，刘琳.急性肾损伤的定义、诊断及治疗[A].临床儿科杂志，2009，27（4）：301-306
12Mehta RL，Kellum JA，Shah SV，et al. Acute renal failure-definition，outcome measures， animal moldels， fluid therapy and informationtechnology needs：the Second International Consensus Conference of theAcute Dialysis Quality Initiative(ADQI) Group [J].Crit Care Med，2007，
35(8)：1837-1843
13Waikar SS，Boncentre JV.Biomarkers for the diagnosis of acute kidneyinjury.Curr Opin Nephrol Hypertens，2007，16：557-564
14Vaidya VS，Ferguson MA，Bonventre JV. Biomarkers of acute kidneyinjury. Annu Rev Pharmacol Toxicol，2008，48：463-493
15Nguyen M T，Decarajan P. Biomarkers for the early detection of acutekidney injury[J].Pediatr Nephrol，2008，23：2151-2157
16Vaidya VS，Ramirez V，Ichimura T，ct al.Urinary kidney injurymolccule-1：a sensitive quantitative biomarker for early detection ofkidney tubular injury[J].Am J Physiol Renal Physiol，2006，290(2)：F517-F529
17Dagher PC，Herget-Rosenthal S，Ruehm SG，et al. Newly developedtechniques to study and diagnose acute renal failure[J].J Am SocNephrol，2003，14(8)：2188-2198
18Parikh CR，Abraham E，Ancukiewicz M，et al.Urine IL-18is an earlydiagnostic marker for acute kidney injury and predicts mortality in theintensive care unit[J].J Am Soc Nephrol，2005，16(10)：3046-3052
19Muramatsu Y，Tsujie M，Kohda Y，et al. Early detection of cysteine richprotein61(CYR61，CCNI)in urine following renal ischemic reperfusioninjury [J].Kidney Int，2002，62(5)：1601-1610
20Du Cheyron D，Daubin C，Poggioli J，et al.Urinary measurement ofNa+/H+exchanger isoform3(NHE3) protein as new marker of tubuleinjury critically ill patients with ARF[J].Am J Kidney Dis，2003，42(3)：497-506
21金霞，王红云.胱抑素C在临床中的运用研究[B].中国健康月刊（学术版），1005-0510（2011）08-0343-021
22戎殳综述，梅长林审校.急性肾损伤定义诊断及治疗进展[A].Chinese Journal of Practical Internal Medicine Nov.2006Vol.26No.21，1005-2194（2006）21-1740-04
23蒋芬，梁馨苓.急性肾损伤临床研究进展[J].实用医学杂志2011年第
27卷第6期：933-935
24张祯，王晓明.血清胱抑素C量血蛋白检测对早期糖尿病损害的意义[J].中国医学检验杂志，2008，29（1）：3
25王鹏飞，沈玲红，何奔.急性肾损伤生物标志物的研究进展[C].J InternMed Concepts Pract2010，Vol.5，No.3：268-272
26Coca SG，Parikh CR.Urinary biomarkers for acute kidney injury:：perspectives on translation. Chin J A Soc Nephrol，2008，4：481-490
27Herget-Rosenthal S，Pietruck F，Volbracht L，et al. Serum cystatin C—asuperior marker of rapidly reduced glomerular filtration afteruninephrectomy in kidney donors compared to creatine [J].Clin Nephrol，2005，64（1）：41-46
28Ichimura TI，Hung CC，Yang SA，et al. Kidney injury molecule-1：atissue and urinary biomarker for nephrotoxicant-induced renal injury[J].Am J Physiol Renal Physiol，2006，286(3)：552-563
29Herget-Rosenthal S，Marggraf G，Husing J，et al. Early detection of acuterenal failure by serum cystatin C [J].Kidney Int，2004，66(3)：1115-1122
30吴亚君，施岚，范亚平，等.尿液西司他丁测定与肾小球损害的相关性研究[J].放射免疫学杂志，2004，17（3）：232-233
31Dharnidharka T，Kwon C，Stevens G，et al. Serum cystatin C is superiorto serum creatinine as a marker of kidney function：A meta-analysis[J].Am J Kidney Disease，2002，40(2)：221-226
32Michael Bennett，：Catherine L Dent，Qing Ma，et al. Urine NGALPredicts Severity of Acute Kidney Injury After Cardiac Surgery：AProspective Study [J]. Clin J Am Soc Nephrol，2008，3(3)：665-669.
33Han WK，Bailly V，Abichandani R，et al. Kidney injury molecule-1(KIM-1)：a novelbiomarker for human renal proximal tubule injury[J].Kidney Int2002，62(1)：237-244
34Zhang Z，Humphreys BD，Boruentre JV. Shedding of the urinarybiomarker kidney injury molecule-1(KIM-1) is regulated by MAPkinases and juxtamembrane region [J].J Am Soe Nephrol，2007，18(10)：2701-2711
35Liangos O，Perianayagam M C, Vaidya, et al.Urinary N-acetyl-beta(D)-glucosaminidase activity and kidney injury molecule-1level areassociated with adverse outcomes in acute renal failure [J].J Am SocNephrol，2007，18(3)：904-912
36Ostermann M，Chang RW.Acute kidney injury in the intensive care unitaccording to RIFLE[J].Crit Care Med，2007,35（8）：1837-1843
37Mishra J，Dent C，Tarabishi R，et al.Neutrophil gelatinase-associatedlipocalin（NGAL） as a biomarker for acute renal injury after cardiacsurgeru[J].Lancet，2005,365（9466）：1231-1238
38Parikh CR，Jani A，Melnikov VY，et al.Urinary interleukin18is a markerof human acute tubular necrosis[J].Am J Kidney Dis，2004,43（3）：405-414
39Washburn KK，Zappitelli M，Arikan AA，et al.Urinary interleukin-18isan acute kidney injury biomarker in crttically ill children[J].Nephrol DialTransplant，2008，23（2）：566-572
40Du Cheyron D，Daubin C，Poggioli J，et al.Urinary measurement of Na＋/H＋exchanger isoform3（NHE-3）protein as new marker of tubuleinjury in critically ill patients with ARF[J].Am J Kidney Dis，2003,42（3）：497-506
41Westhuyzen J，Endre ZH，Reece G，et al.Measurement of tubularenzymuria facilitates early detection of acute renal impairment in theintensive care unit[J].Nephrol Dial Transpant，2003,18（3）：543-551
42Vaikya VS，Ferguson MA，Bonventre JV.Biomarkers of acute kidneyinjury[J].Annu Rev Pharmacol Toxicol，2008,48:463-493
43Rothlein R，Ocerview of leukocyte adhesion.Neurology，1997：49-53
44Rothlein R，Dustin ML，Marlin SD，et al[J].Lnmunol，1986，137：1270-1274