补体C5a受体在单侧输尿管梗阻大鼠肾组织中的表达
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摘要
研究背景
研究显示肾间质病变与慢性肾功能衰竭的进展关系密切,肾间质纤维化几乎是各种肾脏疾病进展到终末期肾衰竭的共同途径和主要病理基础。研究其病理过程和发生机制,寻找延缓和阻断肾间质纤维化新靶标,是目前国内外肾脏病研究的热点方向。
肾间质纤维化的病理过程主要是肾小管-间质损伤,炎症细胞侵入,促进纤维化的因子包括转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、血小板源性生长因子(PDGF)等生成增加,抑制纤维化的因子例如:肝细胞生长因子(HGF)和核心蛋白多糖(Decorin)等生成减少;还有肌成纤维细胞形成和增值,细胞外基质(ECM)积聚的过程。在众多促纤维化的因子中,TGF-β1被认为是最重要的促纤维化因子。很多研究试图通过抑制TGF-β1阻止肾纤维化进展。然而,要将其研究应用于临床尚有很多问题需要解决,例如,目前的方法还很难在人体肾脏产生显著的反义抑制TGF-β1表达的寡义核苷酸来抑制TGF-β1的表达。其次TGF-β1也是一个抗炎细胞因子,长期抑制TGF-β1会诱发炎症,研究发现TGF-β1基因敲除的小鼠可死于多发性炎症。
因此迫切需要寻找防治肾间质纤维化的更好靶点,在抗纤维化的同时不影响机体的正常生理功能。
补体C5是新近发现的致纤维化因子,参与肝、肺等多种器官的纤维化进展,最近研究发现在肾纤维化的进展发挥作用。
Peter Boor等发现在单侧输尿管梗阻(UUO)诱导的小鼠急性肾间质纤维化模型中,C 5基因敲除(应用基因敲除技术敲除某些特定基因使C 5不表达)小鼠组的肾纤维化程度显著低于正常小鼠组,尤其在梗阻早期(第5天到第10天)C 5基因敲除小鼠组在Ⅰ型胶原、纤维连接蛋白、平滑肌肌动蛋白、波形蛋白等纤维化指标上与野生正常小鼠组相比显著降低。由此认为在肾脏疾病中C 5作为新发现的促纤维化因子,有可能成为预防和治疗肾纤维化的潜在靶标。
补体C5a受体(C5aR)是补体系统中的一重要分子,主要表达在髓系来源的细胞的细胞膜上,通过与C5a(补体C5的裂解片段)的结合发挥作用。最近研究发现其在正常人肾脏的近曲小管上皮细胞也有表达,并且参与肾间质损伤的过程。在蛋白尿肾纤维化实验动物模型中肾小球滤过的和肾脏局部产生的补体蛋白在肾脏近曲小管活化后与局部的补体受体结合产生趋化粒细胞因子,趋化巨噬细胞等粒细胞向炎症处游离,而巨噬细胞诱导多种细胞因子的释放等,促进肾损伤的进程。但是关于C5aR在非蛋白尿的肾损伤实验动物模型中的作用及其机制,目前国内尚未见到此方面的报道。Peter Boor首次报道补体C5基因敲除小鼠在单侧输尿管梗阻引起的肾纤维化模型中,肾纤维化程度明显减轻。而UUO模型是非蛋白尿的肾损伤实验动物模型,在UUO模型中C5aR发挥作用的机制是否与其在蛋白尿肾损伤实验动物模型中一样,是否有其他机制在UUO模型肾损伤中发挥作用,还不是很清楚。补体C 5的作用主要是由C 5a(C5的裂解片段,具有活性)与C5aR结合介导,C5aR是否在U U O诱导的肾间质纤维化模型中是否介导补体C 5的致纤维化作用,尚不清楚。
补体系统在肾脏疾病中扮演损伤和保护两种角色,Stefan P.Berger等通过基因敲除的肾炎动物模型的研究发现补体活化的上游成分对机体有有益作用:比如调理作用和清除体内免疫复合物和细胞凋亡等;而下游成分比如C5,则起有害作用。C5抑制剂可以阻断肾小球肾炎的进展,抑制补体下游成分C5和膜攻复合物(MAC)的产生,而保留上游成分对机体的保护作用。如果我们通过应用C5抑制剂或者C5aR受体阻滞剂可以阻断补体C5在肾纤维化中的作用,达到延缓肾纤维化进程,减轻肾纤维化程度,而同时不影响其抗炎作用,这对防治肾纤维化将起到积极作用。
UUO模型诱导的小鼠肾小管间质纤维化,是公认的用于研究急性肾间质纤维化的经典模型,可以迅速达到实验的要求且无蛋白尿,广泛应用于肾间质纤维化的研究。
我们通过结扎单(左)侧输尿管,引起输尿管急性梗阻,诱导急性肾间质纤维化模型来研究C5aR和TGF-β1在肾间质纤维化中组织中的表达,探讨C5aR和TGF-β1在肾间质纤维化中相关关系及可能的作用机制,为临床防治肾纤维化提供新思路,也为进一步解释肾间质纤维化的发生发展机制提供有益参考。
研究目的
1、动态观测单侧输尿管梗阻大鼠肾组织中C5aR和TGF-β1的表达变化,探讨C5aR和TGF-β1在肾纤维化进程中的相关关系;
2、探讨C5aR在肾小管间质纤维化发展中的作用机制。
研究方法
1、造单侧输尿管梗阻模型
将36只雄性SD大鼠随机分为UUO组及假手术组(SOR)组,UUO组行左侧输尿管结扎术,SOR组仅游离左侧输尿管不结扎。分别于术后第5、10、15天处死每组中的6只大鼠,取左肾(梗阻侧)行HE和Masson染色。
2、用免疫组化ELiVision~(TM)plus法检测肾组织C5aR和TGF-β1的表达。
3、用SPSS13.0对结果进行数据分析,本实验为2×3*6的析因设计,行方差分析了解梗阻和时间两处理因素的单独效应、主效应和交互效应,每个指标的组内、组间比较采用单因数方差分析,多重比较若方差齐采用SNK或LSD法,不齐采用DunnettT3或Dunnett C检验,C5aR、TGF-β1免疫组化阳性程度与肾纤维化程度百分比、肾小管间质损伤指数的相关分析采用Spearmen相关系数法分析和偏相关系数法分析C5aR免疫组化阳性程度与肾纤维化程度百分比、肾小管间质损伤指数的相关关系,当P＜0.05时,差异有统计学意义。
结果
1、HE、MASSON染色评估肾纤维化程度
结果模型组第5天远端小管扩张,肾间质轻度水肿,间质内有炎细胞浸润;梗阻第10天近端小管扩张,部分小管上皮细胞变性坏死;第15天可见弥漫的肾小管基底膜增厚皱缩,胶原增多,纤维化明显,随着梗阻时间延长,肾间质纤维化程度不断进展,肾小管损伤指数不断升高,且不同时间点差异有显著统计学意义(P＜0.05)。
2、免疫组化结果显示
C5aR和TGF-β1在假手术组大鼠肾组织中仅有少量表达。模型组大鼠梗阻侧肾组织中C5aR和TGF-β1表达随着肾纤维化程度进展而表达明显增多与假手术组不同时间点相比差异有统计学意义(P＜0.05),而C5aR在梗阻晚期(第10-15天)增多并不明显,梗阻第10天和梗阻第15天相比差异无统计学意义。
结论
C5aR除了在肾间质和远曲小管有表达外,在集合管上皮细胞、肾小球系膜细胞均有表达,C5aR免疫组化阳性强度同肾间质损伤指数、肾间质纤维化程度显著正相关,控制TGF-β1后行偏相关分析仍呈正相关,且随着梗阻时间的延长表达逐渐增多,提示其可能通过一条与TGF-β1的信号通路相平行的通路独立发挥作用,介导补体C 5在肾纤维化中的作用;TGF-β1主要在肾脏近曲小管、远曲小管、集合管上皮细胞和小球系膜细胞表达,且以细胞胞浆表达为主,在SOR组表达微弱,在UUO组随着梗阻时间延长,免疫组化阳性强度逐渐增强,TGF-β1是致肾纤维化的主要因子,在肾纤维化进展中发挥重要作用,通过本实验我们不能排除C5aR也可能作为TGF-β1的上游信号因子在肾间质纤维化早期通过上调TGF-β1发挥致纤维化作用。
Reserch background
The reserch shows that the relation between renal interstitial disease and the progression of chronic kidney failure is closely.Renal interstitial fibrosis is a common feature of progressive renal diseases.It is a very hots pot to study the pathological process and mechanism of renal interstitial disease and search a new target for delaying and blocking the progressive of renal fibrosis.Although most reseachers try to block the progression of renal interstitial fibrosis,there isnot a satisfying result.
The major pathological process of renal interstitial fibrosis includs the injuriing of kidney tubules and interstitial tissue,invading of inflammatory cell,the increasing of transforming growth factor-β1(TGF-β1),Connective Tissues growth factor)(CTGF),platelet-derived growth factor(PDGF)etal,and the decresing of hepatocyte growth factor(HGF),and decorin etal;the increment of myofibroblast;the accumulatio of extracellular matrix(ECM).The TGF-β1 was been known as the most important promoting fibrosis factor.Although most researchers tried to block the progression of Renal interstitial fibrosis by restrainning the TGF-β1,it needs a lot of works to make use of TGF-β1 for clinical patients.Besides the inhibition of TGF-β1 may result in inflammation and lead to dead.
Therefore it is need baddly to look for a better taget for preventing renal fibrosis,and it is donnot influence the normal physiological function,when we want to cure the renal fibbrosis with blocking the target.
Complement 5 is a new factor which can lead to renal fibrosis,it takes part in the progression of the fibrosis of liver,lung etal.
Professor PeterBoor etal detected that the degree of renal fibrosis of C5 knocked out mice is lower than normal mice after the mice were been ligated the left ureter.The collagen,fibronectin,smooth muscle actin,Vimentin and infiltrative macrophages were much lower especical during the nonage of obstruction(from 5~(th) to 10~(th)).So he thinks that C5 may be a new factor for promoting the process of renal fbrosis,and it may been thougt a new target for preventing and curing renal fibrosis.
Complement fragment 5a receptor(C5aR) is an important element in the systerm of Complement,which are been expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages,neutrophils and T cells,and produce its effect by binding with C5a.Recently,it is been detected that C5aR was been expressed on the epithelium of proximal convoluted tubule for normal human,and take part in the progress of renal fibrosis.Protein C Which was be filtrated by glomerular and produced by renal can produce lots of factors which can promote the progrecess of renal fibrosis by binding with C5a in the Experimental Animal Models which was maded by unilateral ureteral obstruction(UUO)and lead to proteinuria.However,The effect and mechanism of C5aR are not clear in the Experimental Animal Models which is nonalbuminuric.PeterBoor etal is the first person who reports that the degree of renal fibrosis was decrease obviously in the C5 knockedout mice following UUO.It is not clear that the role of C5aR in the Experimental Animal Models of UUO.
Complement system play damaging and protecting role in kidney disease.Stefan P.Berger etaldiscovered that the upper stream of complement system plays a benificial effect,and the downstream plays a adverse effect.The inhibitorof C5 can interrupt the progression of brightic and the production of membrane attack complex,doesnot affect the effect of upper stream.If we make use of the inhibitor of C5 and C5aR,we can block the progression of renal fibrosis and relieve the degree of renal fibrosis.It is very positive for the reserch of blocking renal fibrosis.
The Experimental Animal Models which was been made by UUO was been accepted by most reserchers who were studying the mechanism of renal fibrosis.
We are going to reserch the expression of C5aR and TGF-β1 on the mice after UUO and investigate the relation between C5aR and TGF-β1 during the progression of renal fibrosis.We hope we can get a new thread to cure the renal fibrosis and provid some benificial conclusion which can explain the mechanism of renal fibrosis.
Object
1、To study the expression of和TGF-β1 in SD rats with Unilateral Ureteral Obstruction(UUO)and the relation between C5aR和TGF-β1 following UUO.
2、To reserch the role of C5aR during the progreession of renal fibrosis following UUO.
Methods
1、Thirty six male Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group,and UUO group.UUO model was induced by ligating the left ureter of rats.Six rats in each group were sacrificed 5~(th),10~(th),15~(th) days after UUO.
2、Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of C5aR,TGF-beta1 were detected by immunohistochemical staining.
3、The SPSS(version 13.0) software packages were used for the statistical analysis.We use factorial analysis to analyse the results and use correlation and Partial analysis to analysis the correlation between the rate of positive areas of TGF-β1 C5aR and renal tubulointerstitial injury index,All the analyses used two-tailed probability.Given the possible risk of TypeⅠerror,significant results must be considered with caution when P-values are marginally_0.05.
Results
1、At the 5~(th) day,there was renal tubular ectasia,interstitial cells were partialy denaturated in the UUO group.when getting the 10th day,we found necrosis of interstitial cells in renal tubule and an increase of fiber in interstitial substance.While at the 15~(th) day,there was asystematic thickening and shrinkage of renal tubular basement membrane,and the fibrosis was obvious(p＜0.05).Injure exponent of interstitial substance and the extent of nephritic fibrosis in UUO group were much higher than that in SOR group at different time point respectively(p＜0.05).
2、Results of immunohistochemistry indicated that:there was only a little expression of C5aR in nephridial tissue of SOR group's rats.The expression of C5aR in the UUO group at the 5~(th),10~(th),15~(th) day[7.94±2.67,12.39±2.64,13.37±0.98]was much higher than that in SOR group[4.50±2.32,5.69±1.73,4.30±1.45](P＜0.05) respectively.In addition,The C5aR's expression at the 10~(th) day was much higher than that at the 5~(th) day(P＜0.05),while the increase from the 10~(th) day to 15~(th) day was just a little.It is same to be seen on the expressions of TGF-beta1.
Conclusion
C5aR may take part in renal intersititial fibrosis at the early stage.C5aR play a benifitial role in the kidney by upregulating the TGF-beta1 expression and promoting the renal interstitial fibrosis following UUO,or maybe there was another path which directly lead to renal fibrosi with C5aR bonding C5a.
研究显示肾间质病变与慢性肾功能衰竭的进展关系密切,肾间质纤维化几乎是各种肾脏疾病进展到终末期肾衰竭的共同途径和主要病理基础。研究其病理过程和发生机制,寻找延缓和阻断肾间质纤维化新靶标,是目前国内外肾脏病研究的热点方向。
肾间质纤维化的病理过程主要是肾小管-间质损伤,炎症细胞侵入,促进纤维化的因子包括转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、血小板源性生长因子(PDGF)等生成增加,抑制纤维化的因子例如:肝细胞生长因子(HGF)和核心蛋白多糖(Decorin)等生成减少;还有肌成纤维细胞形成和增值,细胞外基质(ECM)积聚的过程。在众多促纤维化的因子中,TGF-β1被认为是最重要的促纤维化因子。很多研究试图通过抑制TGF-β1阻止肾纤维化进展。然而,要将其研究应用于临床尚有很多问题需要解决,例如,目前的方法还很难在人体肾脏产生显著的反义抑制TGF-β1表达的寡义核苷酸来抑制TGF-β1的表达。其次TGF-β1也是一个抗炎细胞因子,长期抑制TGF-β1会诱发炎症,研究发现TGF-β1基因敲除的小鼠可死于多发性炎症。
因此迫切需要寻找防治肾间质纤维化的更好靶点,在抗纤维化的同时不影响机体的正常生理功能。
补体C5是新近发现的致纤维化因子,参与肝、肺等多种器官的纤维化进展,最近研究发现在肾纤维化的进展发挥作用。
Peter Boor等发现在单侧输尿管梗阻(UUO)诱导的小鼠急性肾间质纤维化模型中,C 5基因敲除(应用基因敲除技术敲除某些特定基因使C 5不表达)小鼠组的肾纤维化程度显著低于正常小鼠组,尤其在梗阻早期(第5天到第10天)C 5基因敲除小鼠组在Ⅰ型胶原、纤维连接蛋白、平滑肌肌动蛋白、波形蛋白等纤维化指标上与野生正常小鼠组相比显著降低。由此认为在肾脏疾病中C 5作为新发现的促纤维化因子,有可能成为预防和治疗肾纤维化的潜在靶标。
补体C5a受体(C5aR)是补体系统中的一重要分子,主要表达在髓系来源的细胞的细胞膜上,通过与C5a(补体C5的裂解片段)的结合发挥作用。最近研究发现其在正常人肾脏的近曲小管上皮细胞也有表达,并且参与肾间质损伤的过程。在蛋白尿肾纤维化实验动物模型中肾小球滤过的和肾脏局部产生的补体蛋白在肾脏近曲小管活化后与局部的补体受体结合产生趋化粒细胞因子,趋化巨噬细胞等粒细胞向炎症处游离,而巨噬细胞诱导多种细胞因子的释放等,促进肾损伤的进程。但是关于C5aR在非蛋白尿的肾损伤实验动物模型中的作用及其机制,目前国内尚未见到此方面的报道。Peter Boor首次报道补体C5基因敲除小鼠在单侧输尿管梗阻引起的肾纤维化模型中,肾纤维化程度明显减轻。而UUO模型是非蛋白尿的肾损伤实验动物模型,在UUO模型中C5aR发挥作用的机制是否与其在蛋白尿肾损伤实验动物模型中一样,是否有其他机制在UUO模型肾损伤中发挥作用,还不是很清楚。补体C 5的作用主要是由C 5a(C5的裂解片段,具有活性)与C5aR结合介导,C5aR是否在U U O诱导的肾间质纤维化模型中是否介导补体C 5的致纤维化作用,尚不清楚。
补体系统在肾脏疾病中扮演损伤和保护两种角色,Stefan P.Berger等通过基因敲除的肾炎动物模型的研究发现补体活化的上游成分对机体有有益作用:比如调理作用和清除体内免疫复合物和细胞凋亡等;而下游成分比如C5,则起有害作用。C5抑制剂可以阻断肾小球肾炎的进展,抑制补体下游成分C5和膜攻复合物(MAC)的产生,而保留上游成分对机体的保护作用。如果我们通过应用C5抑制剂或者C5aR受体阻滞剂可以阻断补体C5在肾纤维化中的作用,达到延缓肾纤维化进程,减轻肾纤维化程度,而同时不影响其抗炎作用,这对防治肾纤维化将起到积极作用。
UUO模型诱导的小鼠肾小管间质纤维化,是公认的用于研究急性肾间质纤维化的经典模型,可以迅速达到实验的要求且无蛋白尿,广泛应用于肾间质纤维化的研究。
我们通过结扎单(左)侧输尿管,引起输尿管急性梗阻,诱导急性肾间质纤维化模型来研究C5aR和TGF-β1在肾间质纤维化中组织中的表达,探讨C5aR和TGF-β1在肾间质纤维化中相关关系及可能的作用机制,为临床防治肾纤维化提供新思路,也为进一步解释肾间质纤维化的发生发展机制提供有益参考。
研究目的
1、动态观测单侧输尿管梗阻大鼠肾组织中C5aR和TGF-β1的表达变化,探讨C5aR和TGF-β1在肾纤维化进程中的相关关系;
2、探讨C5aR在肾小管间质纤维化发展中的作用机制。
研究方法
1、造单侧输尿管梗阻模型
将36只雄性SD大鼠随机分为UUO组及假手术组(SOR)组,UUO组行左侧输尿管结扎术,SOR组仅游离左侧输尿管不结扎。分别于术后第5、10、15天处死每组中的6只大鼠,取左肾(梗阻侧)行HE和Masson染色。
2、用免疫组化ELiVision~(TM)plus法检测肾组织C5aR和TGF-β1的表达。
3、用SPSS13.0对结果进行数据分析,本实验为2×3*6的析因设计,行方差分析了解梗阻和时间两处理因素的单独效应、主效应和交互效应,每个指标的组内、组间比较采用单因数方差分析,多重比较若方差齐采用SNK或LSD法,不齐采用DunnettT3或Dunnett C检验,C5aR、TGF-β1免疫组化阳性程度与肾纤维化程度百分比、肾小管间质损伤指数的相关分析采用Spearmen相关系数法分析和偏相关系数法分析C5aR免疫组化阳性程度与肾纤维化程度百分比、肾小管间质损伤指数的相关关系,当P＜0.05时,差异有统计学意义。
结果
1、HE、MASSON染色评估肾纤维化程度
结果模型组第5天远端小管扩张,肾间质轻度水肿,间质内有炎细胞浸润;梗阻第10天近端小管扩张,部分小管上皮细胞变性坏死;第15天可见弥漫的肾小管基底膜增厚皱缩,胶原增多,纤维化明显,随着梗阻时间延长,肾间质纤维化程度不断进展,肾小管损伤指数不断升高,且不同时间点差异有显著统计学意义(P＜0.05)。
2、免疫组化结果显示
C5aR和TGF-β1在假手术组大鼠肾组织中仅有少量表达。模型组大鼠梗阻侧肾组织中C5aR和TGF-β1表达随着肾纤维化程度进展而表达明显增多与假手术组不同时间点相比差异有统计学意义(P＜0.05),而C5aR在梗阻晚期(第10-15天)增多并不明显,梗阻第10天和梗阻第15天相比差异无统计学意义。
结论
C5aR除了在肾间质和远曲小管有表达外,在集合管上皮细胞、肾小球系膜细胞均有表达,C5aR免疫组化阳性强度同肾间质损伤指数、肾间质纤维化程度显著正相关,控制TGF-β1后行偏相关分析仍呈正相关,且随着梗阻时间的延长表达逐渐增多,提示其可能通过一条与TGF-β1的信号通路相平行的通路独立发挥作用,介导补体C 5在肾纤维化中的作用;TGF-β1主要在肾脏近曲小管、远曲小管、集合管上皮细胞和小球系膜细胞表达,且以细胞胞浆表达为主,在SOR组表达微弱,在UUO组随着梗阻时间延长,免疫组化阳性强度逐渐增强,TGF-β1是致肾纤维化的主要因子,在肾纤维化进展中发挥重要作用,通过本实验我们不能排除C5aR也可能作为TGF-β1的上游信号因子在肾间质纤维化早期通过上调TGF-β1发挥致纤维化作用。
Reserch background
The reserch shows that the relation between renal interstitial disease and the progression of chronic kidney failure is closely.Renal interstitial fibrosis is a common feature of progressive renal diseases.It is a very hots pot to study the pathological process and mechanism of renal interstitial disease and search a new target for delaying and blocking the progressive of renal fibrosis.Although most reseachers try to block the progression of renal interstitial fibrosis,there isnot a satisfying result.
The major pathological process of renal interstitial fibrosis includs the injuriing of kidney tubules and interstitial tissue,invading of inflammatory cell,the increasing of transforming growth factor-β1(TGF-β1),Connective Tissues growth factor)(CTGF),platelet-derived growth factor(PDGF)etal,and the decresing of hepatocyte growth factor(HGF),and decorin etal;the increment of myofibroblast;the accumulatio of extracellular matrix(ECM).The TGF-β1 was been known as the most important promoting fibrosis factor.Although most researchers tried to block the progression of Renal interstitial fibrosis by restrainning the TGF-β1,it needs a lot of works to make use of TGF-β1 for clinical patients.Besides the inhibition of TGF-β1 may result in inflammation and lead to dead.
Therefore it is need baddly to look for a better taget for preventing renal fibrosis,and it is donnot influence the normal physiological function,when we want to cure the renal fibbrosis with blocking the target.
Complement 5 is a new factor which can lead to renal fibrosis,it takes part in the progression of the fibrosis of liver,lung etal.
Professor PeterBoor etal detected that the degree of renal fibrosis of C5 knocked out mice is lower than normal mice after the mice were been ligated the left ureter.The collagen,fibronectin,smooth muscle actin,Vimentin and infiltrative macrophages were much lower especical during the nonage of obstruction(from 5~(th) to 10~(th)).So he thinks that C5 may be a new factor for promoting the process of renal fbrosis,and it may been thougt a new target for preventing and curing renal fibrosis.
Complement fragment 5a receptor(C5aR) is an important element in the systerm of Complement,which are been expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages,neutrophils and T cells,and produce its effect by binding with C5a.Recently,it is been detected that C5aR was been expressed on the epithelium of proximal convoluted tubule for normal human,and take part in the progress of renal fibrosis.Protein C Which was be filtrated by glomerular and produced by renal can produce lots of factors which can promote the progrecess of renal fibrosis by binding with C5a in the Experimental Animal Models which was maded by unilateral ureteral obstruction(UUO)and lead to proteinuria.However,The effect and mechanism of C5aR are not clear in the Experimental Animal Models which is nonalbuminuric.PeterBoor etal is the first person who reports that the degree of renal fibrosis was decrease obviously in the C5 knockedout mice following UUO.It is not clear that the role of C5aR in the Experimental Animal Models of UUO.
Complement system play damaging and protecting role in kidney disease.Stefan P.Berger etaldiscovered that the upper stream of complement system plays a benificial effect,and the downstream plays a adverse effect.The inhibitorof C5 can interrupt the progression of brightic and the production of membrane attack complex,doesnot affect the effect of upper stream.If we make use of the inhibitor of C5 and C5aR,we can block the progression of renal fibrosis and relieve the degree of renal fibrosis.It is very positive for the reserch of blocking renal fibrosis.
The Experimental Animal Models which was been made by UUO was been accepted by most reserchers who were studying the mechanism of renal fibrosis.
We are going to reserch the expression of C5aR and TGF-β1 on the mice after UUO and investigate the relation between C5aR and TGF-β1 during the progression of renal fibrosis.We hope we can get a new thread to cure the renal fibrosis and provid some benificial conclusion which can explain the mechanism of renal fibrosis.
Object
1、To study the expression of和TGF-β1 in SD rats with Unilateral Ureteral Obstruction(UUO)and the relation between C5aR和TGF-β1 following UUO.
2、To reserch the role of C5aR during the progreession of renal fibrosis following UUO.
Methods
1、Thirty six male Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group,and UUO group.UUO model was induced by ligating the left ureter of rats.Six rats in each group were sacrificed 5~(th),10~(th),15~(th) days after UUO.
2、Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of C5aR,TGF-beta1 were detected by immunohistochemical staining.
3、The SPSS(version 13.0) software packages were used for the statistical analysis.We use factorial analysis to analyse the results and use correlation and Partial analysis to analysis the correlation between the rate of positive areas of TGF-β1 C5aR and renal tubulointerstitial injury index,All the analyses used two-tailed probability.Given the possible risk of TypeⅠerror,significant results must be considered with caution when P-values are marginally_0.05.
Results
1、At the 5~(th) day,there was renal tubular ectasia,interstitial cells were partialy denaturated in the UUO group.when getting the 10th day,we found necrosis of interstitial cells in renal tubule and an increase of fiber in interstitial substance.While at the 15~(th) day,there was asystematic thickening and shrinkage of renal tubular basement membrane,and the fibrosis was obvious(p＜0.05).Injure exponent of interstitial substance and the extent of nephritic fibrosis in UUO group were much higher than that in SOR group at different time point respectively(p＜0.05).
2、Results of immunohistochemistry indicated that:there was only a little expression of C5aR in nephridial tissue of SOR group's rats.The expression of C5aR in the UUO group at the 5~(th),10~(th),15~(th) day[7.94±2.67,12.39±2.64,13.37±0.98]was much higher than that in SOR group[4.50±2.32,5.69±1.73,4.30±1.45](P＜0.05) respectively.In addition,The C5aR's expression at the 10~(th) day was much higher than that at the 5~(th) day(P＜0.05),while the increase from the 10~(th) day to 15~(th) day was just a little.It is same to be seen on the expressions of TGF-beta1.
Conclusion
C5aR may take part in renal intersititial fibrosis at the early stage.C5aR play a benifitial role in the kidney by upregulating the TGF-beta1 expression and promoting the renal interstitial fibrosis following UUO,or maybe there was another path which directly lead to renal fibrosi with C5aR bonding C5a.
引文
1 Yang J ,Liu Y.Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol[J].2001 ,159(4) :1465 -1475.
2 Ma LJ, Jha S,Ling H, et al.Divergent effects of low versus highdose anti-TGF-beta antibody in puromycin aminonucleoside nephropa in rats.Kidney Int[J].2004, 65(1): 106 -115.
3 PeterBoor,Andrzej Konieczny,Luigi Villa,et al.Complement C5 mediates experimental tubulointerstitial fibrosis J Am Soc Nephrol [J].2007,18(5):1508-15.
4 Nangaku M, Pippin J, Couser WG: Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome.J Am Soc Nephrol[J].1999.10(11): 2323-2331.
5 Nangaku M, Pippin J, Couser WG: C6 mediates chronic progression of tubulointerstitial damage in rats with remnant kidneys[J].J Am Soc Nephrol 2002.13(4): 928-936.
6 Wilmer WA, Kaumaya PT, Ember JA, Cosio FG.Receptors for theanaphylatoxin C5a (CD88) on human mesangial cells.J Immunol[J].1998; 160(11):5646-52
7 Zahedi R, Braun M, Wetsel RA,et al.The C5a receptor is expressed by human renal proximal tubular epithelial cells[J].Clin Exp Immunol.2000;121(2):226-233.
8 PN Monk,Scola AM,Madala Ret al.Function, structure and therapeutic potential of complement C5a receptors British Journal of Pharmacology[J], 2007,152(4):429-48
9 齐洁,朱锡华.C5a的粘附分子调节和免疫调节作用.国外医学.免疫学分册,1997;20(2):61-63
10 Stefan P.Complement and the kidney:What the nephrologist needs to know in 2006? Nephrol Dial Transplant[J].2005.20(12):2613-2619
11 薛痕,樊均明陈亮等.大鼠肾间质纤维化动物模型的实验研究.四川动物,2004,23(1):16-20
12 Either,Frank Floege,Jurgenl.Novel insights into renalfibrosis[J]Current Opinion in Nephrology & Hypertension[J].2003,12(3):2227-2321
13 Ember JA,Sanderson SD,Hugli TE,.Induction of interleukin-8 synthesis from monocytes by human C5a anaphylatoxin.Am J Pathol[J].1994,144(2):393-403.
14 Montz H,Koch KC,Zierz R,et al.The role of C5a in interleukin-6 production induced by lipopolysaccharide or interleukin-1.Immunology[J].1991,74(3):373-9.
15 Foreman,K.Vaporciyan AA,Bonish,BK.et al..C5a-induced expression of P-selectin in endothelial cells.[J].Clin.Invest.1994,94(3):1147-55.
16 Wittmann M,Zwirner J,Larsson VA et al.C5a suppresses the production of IL-12 by IFN-gamma-primed and lipopolysaccharide-challenged human monocytes.J Immunol[J].1999,162(11):6763-9.
17 Daveau M,Benard M,Scotte M,et al.Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat.J Immunol[J].2004,173(5):3418-24.
18 O'Barr SA,Caguioa J,Gruol D et al.Neuronal expression of a functional receptor for the C5a complement activation fragment.J Immunol.[J].2001,166(6):4154-62.
19 Fayyazzi A,Scheel O,Werfel T et al.The C5a receptor is expressed in renal proximal tubular but not in pulmonary or hepatic epithelial cells.Immunology [J]2000,99(1):38-45.
20 R.ZAHEDI,M.BRAUN*,R.A.WETSEL,et al.The C5a receptor is expressed by human renal proximal tubularepithelial cells.Clin Exp Immunol[J].2000,121(2):226-233.
21 Abe K,Miyazaki M,Koji T,et al Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization.Kidney Int[J].2001,60(1):137-46.
22 Khan TN,Sinniah R:Role of complement in renal tubular damage.Histopathology[J].1995,26(4):351-356.
23 Hillebrandt S,Wasmuth HE,Weiskirchen R,etal.Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans.NatGenet[J].2005,37(8):835-843.
24 杨旭,何平,李德天等骨形念发生蛋白7在单侧输尿管梗阻大鼠肾组织中的表达.中国医科大学学报[J].2007,36(5):535-538.
25 高红宇,邵菊芳,何晓峰等苦参素对单侧输尿管梗阻大鼠肾脏TGF-β1/Smad3表达的影响.中国现代医学杂志[J].2006,16(20):3094-104.
26 刘建社,张春.朱忠华等.结缔组织生长因子在单侧输尿管梗阻大鼠肾脏中的表达及其意义.中国病理生理杂志[J].2007,23(9):1786-05.
27 Addis-Lieser E,Kohl J,Chiaramonte MG:Opposing regulatory roles of complement factor 5 in the development of bleomycin-induced pulmonary fibrosis.J Immunol.2005,175(3):1894-1902.
2 Ma LJ, Jha S,Ling H, et al.Divergent effects of low versus highdose anti-TGF-beta antibody in puromycin aminonucleoside nephropa in rats.Kidney Int[J].2004, 65(1): 106 -115.
3 PeterBoor,Andrzej Konieczny,Luigi Villa,et al.Complement C5 mediates experimental tubulointerstitial fibrosis J Am Soc Nephrol [J].2007,18(5):1508-15.
4 Nangaku M, Pippin J, Couser WG: Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome.J Am Soc Nephrol[J].1999.10(11): 2323-2331.
5 Nangaku M, Pippin J, Couser WG: C6 mediates chronic progression of tubulointerstitial damage in rats with remnant kidneys[J].J Am Soc Nephrol 2002.13(4): 928-936.
6 Wilmer WA, Kaumaya PT, Ember JA, Cosio FG.Receptors for theanaphylatoxin C5a (CD88) on human mesangial cells.J Immunol[J].1998; 160(11):5646-52
7 Zahedi R, Braun M, Wetsel RA,et al.The C5a receptor is expressed by human renal proximal tubular epithelial cells[J].Clin Exp Immunol.2000;121(2):226-233.
8 PN Monk,Scola AM,Madala Ret al.Function, structure and therapeutic potential of complement C5a receptors British Journal of Pharmacology[J], 2007,152(4):429-48
9 齐洁,朱锡华.C5a的粘附分子调节和免疫调节作用.国外医学.免疫学分册,1997;20(2):61-63
10 Stefan P.Complement and the kidney:What the nephrologist needs to know in 2006? Nephrol Dial Transplant[J].2005.20(12):2613-2619
11 薛痕,樊均明陈亮等.大鼠肾间质纤维化动物模型的实验研究.四川动物,2004,23(1):16-20
12 Either,Frank Floege,Jurgenl.Novel insights into renalfibrosis[J]Current Opinion in Nephrology & Hypertension[J].2003,12(3):2227-2321
13 Ember JA,Sanderson SD,Hugli TE,.Induction of interleukin-8 synthesis from monocytes by human C5a anaphylatoxin.Am J Pathol[J].1994,144(2):393-403.
14 Montz H,Koch KC,Zierz R,et al.The role of C5a in interleukin-6 production induced by lipopolysaccharide or interleukin-1.Immunology[J].1991,74(3):373-9.
15 Foreman,K.Vaporciyan AA,Bonish,BK.et al..C5a-induced expression of P-selectin in endothelial cells.[J].Clin.Invest.1994,94(3):1147-55.
16 Wittmann M,Zwirner J,Larsson VA et al.C5a suppresses the production of IL-12 by IFN-gamma-primed and lipopolysaccharide-challenged human monocytes.J Immunol[J].1999,162(11):6763-9.
17 Daveau M,Benard M,Scotte M,et al.Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat.J Immunol[J].2004,173(5):3418-24.
18 O'Barr SA,Caguioa J,Gruol D et al.Neuronal expression of a functional receptor for the C5a complement activation fragment.J Immunol.[J].2001,166(6):4154-62.
19 Fayyazzi A,Scheel O,Werfel T et al.The C5a receptor is expressed in renal proximal tubular but not in pulmonary or hepatic epithelial cells.Immunology [J]2000,99(1):38-45.
20 R.ZAHEDI,M.BRAUN*,R.A.WETSEL,et al.The C5a receptor is expressed by human renal proximal tubularepithelial cells.Clin Exp Immunol[J].2000,121(2):226-233.
21 Abe K,Miyazaki M,Koji T,et al Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization.Kidney Int[J].2001,60(1):137-46.
22 Khan TN,Sinniah R:Role of complement in renal tubular damage.Histopathology[J].1995,26(4):351-356.
23 Hillebrandt S,Wasmuth HE,Weiskirchen R,etal.Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans.NatGenet[J].2005,37(8):835-843.
24 杨旭,何平,李德天等骨形念发生蛋白7在单侧输尿管梗阻大鼠肾组织中的表达.中国医科大学学报[J].2007,36(5):535-538.
25 高红宇,邵菊芳,何晓峰等苦参素对单侧输尿管梗阻大鼠肾脏TGF-β1/Smad3表达的影响.中国现代医学杂志[J].2006,16(20):3094-104.
26 刘建社,张春.朱忠华等.结缔组织生长因子在单侧输尿管梗阻大鼠肾脏中的表达及其意义.中国病理生理杂志[J].2007,23(9):1786-05.
27 Addis-Lieser E,Kohl J,Chiaramonte MG:Opposing regulatory roles of complement factor 5 in the development of bleomycin-induced pulmonary fibrosis.J Immunol.2005,175(3):1894-1902.