NGAL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,寻找延缓和阻断肾纤维化新靶标,是目前国内外肾脏病研究的热点方向。多种细胞因子和生物活性物质参与肾间质纤维化的发生发生发展过程,各调节因子通过生物信号传导机制联系起来,形成完善的调节反馈系统,调节机体的病理生理过程。
肾间质纤维化的发生、发展过程较为复杂,主要包括成纤维细胞活化、增殖及表型转化,转变为分泌α-平滑肌肌动蛋白的肌成纤维细胞,生成细胞外基质(extracelluarmatrix, ECM)的能力明显增强,在多种细胞因子作用、炎症细胞浸润等因素调节下,造成ECM生成/降解平衡失调,ECM大量积聚,从而促进肾间质纤维化的发展。在该病理过程中,有多种细胞因子参与,其中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,抑制纤维化的因子包括：肝细胞生长因子(HGF)、核心蛋白多糖(Decorin)、骨形态形成蛋白-7(BMP-7)等。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反义抑制TGF-β1表达的寡义核苷酸来抑制TGF-β1的表达；且TGF-β1也是一种抗炎因子,长期抑制TGF-β1易诱发炎症反应,研究发现TGF-β1基因敲除的小鼠可死于多发性炎症。因此寻找抑制肾间质的纤维化进程的更特异性的药物作用靶点,在治疗肾脏纤维化中具有重要意义。
基质金属蛋白酶(matrix metalloproteinases, MMPs)是降解ECM的重要酶系,MMPs主要有两方面功能,一是降解全部ECM成分,二是激活其他MMPs,形成级联效应。MMPs均以酶原的形式分泌到ECM中,在激活剂作用下脱去前肽产生具有生物活性的MMPs发挥生物效能。基质金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase, TIMPs)可抑制MMPs酶原及酶活性,对调节ECM形成／降解具有重要作用。肾小管间质纤维化主要是Ⅰ、Ⅱ、Ⅲ型间质胶原及Ⅳ型膜胶原、糖蛋白和蛋白多糖的堆积,MMPs/TIMPs平衡失调肾间质纤维化发病的关键,而MMP-9/TIMP-1为其中最重要的一对。TIMP-1可与MMP-9前体(pro-MMP-9)形成稳定的复合体,阻碍pro-MMP-9的自我激活；同时TIMP-1抑制已活化的MMP-9的活性,直接与活化的MMP-9 Zn2+活性中心按1：1比例发生不可逆结合,阻断或抑制MMP-9活性,而MMP-9活性高低直接影响肾间质纤维化的进程。在不同病因所致的肾间质纤维化病程发展中均存在MMPs/TIMPs的功能紊乱。不同实验方法观察可有不同的结果,但有一种共同趋势,即TIMPs高表达,和(或)MMPs的活性下降。寻找MMPs激活剂或表达促进剂或者TIMPs的抑制剂,有可能成为预防和治疗肾间质纤维化的新方法。
中性粒细胞明胶酶相关脂质运载蛋白,英文名称为Neutrophil gelatinase-associated lipocalin(NGAL),又称为lipocalin-2,是lipocalin家族新成员,是1993年K Jeldsen在中性粒细胞中发现的一种小分子量分泌性蛋白。NGAL除具有lipocalin家族共有疏水基结构外,尚存在一游离的巯基,可与MMP-9前体等其他分子结合,为其发挥特殊的生物学作用奠定了基础。NGAL在人体表达于多种组织器官,如：支气管、胃、小肠、肾脏、前列腺、胸腺等。NGAL具有参与炎症趋化与免疫应答反应；参与肾小管上皮细胞的形成、修复；参与肿瘤的发生发展等多种生物学作用。
研究发现NGAL对调节MMP-9/TIMP-1具有一定作用。NGAL可结合于MMP-9上保护MMP-9不被降解。一般情况下,MMP-9是以Pro-MMP-9形式贮存于细胞内,分泌到细胞外之后,只有切去N端的一段序列,MMP-9才表现活性。通常,Pro-MMP-9在细胞内主要以四种方式存在：单体、同源二聚体、NGAL与Pro-MMP-9以分子间二硫键结合形成的异源二聚体(NGAL/Pro-MMP-9)以及此异源二聚体与金属蛋白酶组织抑制剂-1(TIMP-1)形成的三元复合物NGAL/ Pro-MMP-9/TIMP-1。在这种三元复合物中,Pro-MMP-9仍可被激活为有活性的MMP-9,但活性会有所降低。然而当此三元复合物中的TIMP-1通过它的游离N-端结构域去结合和抑制其它的MMPs时,又可形成NGAL/Pro-MMP-9/ TIMP-1/MMPs四元复合物。而当这种四元复合物被活化时,Pro-MMP-9被激活成MMP-9的活性反而比三元复合物时的活性高许多倍。很显然NGAL明显具有调节这些复合物的能力,甚至从某种程度上来讲,NGAL具有抵消复合物中TIMP-1抑制MMP-9活性的作用。故通过探讨NGAL在肾间质纤维化模型中表达情况及与,对于探索其在肾间质纤维化中的作用机制及为治疗肾间质纤维化可能提供新思路。
研究发现急性药物相关性肾炎患者肾组织中NGAL在近端小管的表达较正常人增强,NGAL表达量与间质-小管损害呈负相关,说明NGAL在急性肾小管间质损伤中起到一定保护作用,但具体机制尚不明确,推测NGAL可能在肾间质纤维化中发挥一定作用。Jaya Mishra在急性小鼠急性缺血-再灌注损伤模型中发现,予NGAL治疗可显著减少小管细胞组织病理学损伤,增加近端小管数目,推测NGAL可能诱导肾间质细胞向小管上皮细胞转换而诱导肾小管上皮细胞再生。
目前国内外对NGAL研究多集中于其在乳腺癌等肿瘤发生机制中的作用及在肾脏急性缺血-再灌注损伤、肾移植及心肺分流术等多种因素诱发的急性肾功能损害的早期检测指标等。对于NGAL在肾间质纤维化中的作用研究较少,以上研究均证明NGAL在肾间质纤维化过程中可能起到一定的调节作用,故探讨NGAL在肾间质纤维化模型中表达情况及作用,对于探索其在肾间质纤维化中的作用机制及为治疗肾间质纤维化可能提供新思路。
研究目的
1、动态观测单侧输尿管梗阻大鼠梗阻侧肾组织NGAL、MMP-9及TIMP-1的表达变化,探讨NGAL与MMP-9、TIMP-1在肾纤维化发展过程中的相关关系;
2、探讨NGAL在肾小管间质纤维化发展中的作用机制。
研究方法
1、制作单侧输尿管梗阻模型
将48只雄性SD大鼠随机分为单侧输尿管梗阻组(UUO)及假手术组(SOR),UUO组行左侧输尿管结扎术,SOR组仅游离左侧输尿管不结扎。术后第1、4、7、14天分别处死每组中的6只大鼠,取左肾(梗阻侧肾脏)行HE和Masson染色。
2、用免疫组化ELiVisionTMplus法检测肾组织NGAL、MMP-9、TIMP-1及FN的表达情况。
3、用SPSS13.0对结果进行数据分析。
结果
1、HE、MASSON染色评估肾纤维化程度
UUO组第1天远端小管无扩张,肾间质无明显水肿；术后第4天远端小管扩张,肾间质轻度水肿,间质内散在淋巴细胞、巨噬细胞浸润；术后第7天远端小管扩张明显,肾间质明显水肿,近端小管出现扩张,炎性细胞浸润进行性加重并伴有纤维细胞增生；术后第14天可见肾小管、集合管扩张呈囊状,部分上皮细胞变性、坏死,肾小管结构破坏,管腔塌陷,炎性细胞浸润。
2、FN免疫组化结果
SOR组大鼠FN在肾小球、肾小管基底膜及肾间质有少许表达,UUO组随
Reserch background
The reserch shows that there is close relation between renal interstitial disease and the progression of chronic kidney failure. Renal interstitial fibrosis is a common feature of progressive renal diseases. It is a very hot pot to study the pathological process and mechanism of renal interstitial disease and search a new target for delaying and blocking renal the progression of renal fibrosis. A great many cytokines and biotic activators participate the genesis and development of renal interstitial fibrosis. All regulatory factors were connected through the biological signal-transducting mechanism, forming the consummate regulatory feedback system to regulate the pathological and physiological processes of organism.
The generation and development of renal interstitial fibrosis are complicated. The main course include the fibroblast's activation, generation and phenotype transition, making it transform into myofibroblast, which can secrete a-smooth muscel actin. So that the generation of extracellular matrix(ECM) increaes, and the balance of ECM's production and degradation collapse with the regulation of mutiple cytokines and inflammatory cell infiltration etc, resulting in the accumulation of ECM and the progression of renal interstitial fibrosis. There are multiple cytokines participating the pathological process, including the promoting factors such as transforming growth factor beta-1(TGF-β1), connective tissue growth factor(CTGF), platelet-derived growth factor(PDGF) and the inhibiting factors like hepatocyte growth factor(HGF), decrin, and bone morphogenetic protein-7(BMP-7) etc. TGF-β1 is considered as the most important promoting fibrosis factor of the mutiple promoting cytokines, and most researchers tried to block the progression of Renal interstitial fibrosis by restrainning the TGF-β1. But 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 death. So it's important to find new targets for supressing the development of RIF without hurting the physiologic function of organism.
Matrix metalloproteinases(MMPs) are very important enzymes degrding ECM. MMPs'functions include ECM degration and activating other MMPs causing cascade effects. MMPs were secreted into ECM in form of proenzyme and turn into bioactive MMPs educing biological activity by taking off propeptide with reactivator's effect. Tissue inhibitors of metalloproteinase(TIMPs) can restrain proenzyme and enzyme activity of MMPs and play important roles in regulating the production and degradation of ECM. Renal interstitial fibrosis is mainly caused by accumulation interstitial collagen of typeⅠ、Ⅱ、ⅢandⅣ, glucoprotein and proteoglycan. Imbalance of MMPs and TIMPs is the key point of progression of renal interstitial fibrosis, while MMP-9 and TIMP-1 is the most important pair. TIMP-1 can combine MMP-9 precurosor turning into stable complex and hinder its self-activation. Meanwehile TIMP-1 can retrain activated MMP-9's activity by irreversibly combining its active center with Zn2+by ratio of one by one. However the activity of MMP-9 directly influences the progression of renal intersitial fibrosis. There are functional disorders of MMPs/TIMPs in the progression of renal interstitial fibrosis caused by various origins of diseases. There might be different outcomes using different methods, but there is a common tendency which is the hypo-expression of the TIMPs with or without the lower activity of MMPs. Therefore it is demanded to look for better activators for MMPs or restrainers for TIMPs which might be a new method to prevent and cure renal fibrosis.
Neutrophil gelatinase-associated lipocalin, short for NGAL, also called lipocalin 2, is a new member of the lipocalin family, which was found as a scecreted protein from neutrophilic leukocyte by K jeldsen in 1993. Besides the common structure of hydrophobic group that the lipocalin family have, it still has the nomadic hydrosulfide group, which can combine MMP-9 precurosor and other molecules, establishing foundation for its special biological contribution. NGAL protein expressed in many organs in human body, such as bronchus, stomach, small intestinum, kidney, prostate and thymas gland. NGAL protein can educe its multiple biological effects by participating immune response reaction, the formation and reparation of renal tubular epithelial cells, and generation and progression of tumors.
It is discovered that NGAL protein plays roles in regulating MMP-9 and TIMP-1.NGAL can combine MMP-9 protecting it from degradation. MMP-9 protein is ususlly stored in cells in form of MMP-9 precurosor, and activated after the series of N-apical being cut when secreted into extracelluar enviroment. MMP-9 precurosor usually exsit in four forms:monomer, homodimer, heterodimer of NGAL protein and MMP-9 precurosor connected by intermolecular disulfide bond, and the ternary complex combined with TIMP-1 by heterodimer. MMP-9 precurosor could still be activated in the ternary complex but get lower activity compared with the monomer type. However TIMP-1 of the ternary complex can combine other MMPs protein by its nomadic N apical structural domain, turning into quaternary complex. When MMP-9 precurosor of the quaternary complex is activated, it has much more activity than that of the ternary one. Thus NGAL protein apparently has the ability of regulating the complex mentioned above, which can counteract the supression of TIMP-1 on MMP-9's activity. It might be useful in obseving NGAL's expression in the model of renal interstitial fibrosis to investigate its mechanism of action in RIF and look for new ideas of curing RIF.
Researchers found that the expression of NGAL protein strenthened in proximal tubules in the patients with acute drug induced interstitial nephritis compared with that of normal persons. The expression was negative correlated with the damage of interstitium, which meant that NGAL educed protective effects in the actue interstitial damage, but the spicific mechanism hadn't been identified, speculating that NGAL might be effective in the RIF progression. Jaya Mishra found that NGAL therapy could decrease the pathological damage in tubular epithelial cells significantly and the proximal tubules increased by creating the acute ischemic-reperfusion injury model of mouse, speculating that NGAL might induce interstial cells transforming into epithelial cells, contributing to the rebirth of renal epithelial cells.
The research of NGAL protein at present mostly focus on its effect on the mechanism of tumors such as breast cancer and etc, and as an early detecting cytokine in the acute renal failure caused by renal ischamic-reperfusion injury, kidney transplantation, cardiopulmonary bypass and etc. There are few reaseaches on its effects in renal interstitial fibrosis, however the investigations mentioned above proved that NGAL might play roles in the progression in RIF. So it's necessary to investigate its expression in RIF model and find new methods to prevent and cure RIF.
Objective
1. To observe the expression of NGAL, MMP-9 and TIMP-1 in renal tissues of rats following unilateral uretral obstuction(UUO), and investigate the correlation of cytokines above in the progression of renal interstitial fibrosis.
2. To investigate the mechanism of action for NGAL in the progression of RIF.
Methods
1. Forty-eight male Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group and an unilateral uretral obstruction group. UUO model was induced by ligating the left ureter of rats. Six rats in each group were sacrificed on the 1st,4th,7th and 14th day after UUO.
2、Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of NGAL, MMP-9, TIMP-1 and FN were detected by immunohistochemical staining of ELiVisionTMplus.
3、Analysis of data with SPSS 13.0 software.
Results
1. On the 1st day, there was little expasion in the distal tubules and little edema in the interstitium. On the 4th day there was renal tubular ectasia, scattered leukocyte and macrophage, interstitial cells were partialy denaturated in the UUO group. When the 7th day came, there was severe edema in interstitium and more infiltration of inflammatory cells and proliferation of fibrocyte. necrosis of interstitial cells in renal tubule and an increase of fiber in interstitial substance. Till the 14th day, there was asystematic thickening and shrinkage of renal tubular basement membrane, and the fibrosis was obvious. 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 for FN indicated that:there was only a little expression of FN in renal glomeruli, tubular basement membrane and renal interstitium. While in the UUO group the expression of FN became higher and higher as obstructed time went(P<0.05). And the positive degree was much higher in UUO group than that of SOR group at each time point respectively(P<0.05).
3. NGAL expressed in the proximal tubules in renal tissue in SOR group. Its expression expanded and strengthened in the early stage of RIF(on the 1st and 4th day after UUO), however it decreased as time went(on the 7th and 14th day), still higher than that of SOR group(P<0.05).
4. MMP-9 expressed in the proximal tubular epithelial cells in renal tissue in SOR group. The expression of MMP-9 strengthened in the UUO group, rising in the early stage(from 1st to 7th day) and decreasing after 7th day(P<0.05).
5. There was little expression of TIMP-1 in SOR group. After obstruction the expression of TIMP-1 gradually increased as time went, and the positive degree was higher in the UUO group than that of SOR group(P<0.05).
Conclusion
The expression of NGAL increased on the early stage after obstruction indicated that it played roles in the early stage of renal interstitial fibrosis. There was apparent. negative correlation between NGAL positive degree and the injury index of interstitium and fibrosis degree meaning that it might restrain the progression of RIF. There was positive correlation between NGAL and the ratio of MMP-9 and TIMP-1 indicating that NGAL delayed the progression of RIF by regulating the balance of MMP-9/TIMP-1.
研究显示肾间质病变与慢性肾功能衰竭进展关系密切,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,寻找延缓和阻断肾纤维化新靶标,是目前国内外肾脏病研究的热点方向。多种细胞因子和生物活性物质参与肾间质纤维化的发生发生发展过程,各调节因子通过生物信号传导机制联系起来,形成完善的调节反馈系统,调节机体的病理生理过程。
肾间质纤维化的发生、发展过程较为复杂,主要包括成纤维细胞活化、增殖及表型转化,转变为分泌α-平滑肌肌动蛋白的肌成纤维细胞,生成细胞外基质(extracelluarmatrix, ECM)的能力明显增强,在多种细胞因子作用、炎症细胞浸润等因素调节下,造成ECM生成/降解平衡失调,ECM大量积聚,从而促进肾间质纤维化的发展。在该病理过程中,有多种细胞因子参与,其中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,抑制纤维化的因子包括：肝细胞生长因子(HGF)、核心蛋白多糖(Decorin)、骨形态形成蛋白-7(BMP-7)等。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反义抑制TGF-β1表达的寡义核苷酸来抑制TGF-β1的表达；且TGF-β1也是一种抗炎因子,长期抑制TGF-β1易诱发炎症反应,研究发现TGF-β1基因敲除的小鼠可死于多发性炎症。因此寻找抑制肾间质的纤维化进程的更特异性的药物作用靶点,在治疗肾脏纤维化中具有重要意义。
基质金属蛋白酶(matrix metalloproteinases, MMPs)是降解ECM的重要酶系,MMPs主要有两方面功能,一是降解全部ECM成分,二是激活其他MMPs,形成级联效应。MMPs均以酶原的形式分泌到ECM中,在激活剂作用下脱去前肽产生具有生物活性的MMPs发挥生物效能。基质金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase, TIMPs)可抑制MMPs酶原及酶活性,对调节ECM形成／降解具有重要作用。肾小管间质纤维化主要是Ⅰ、Ⅱ、Ⅲ型间质胶原及Ⅳ型膜胶原、糖蛋白和蛋白多糖的堆积,MMPs/TIMPs平衡失调肾间质纤维化发病的关键,而MMP-9/TIMP-1为其中最重要的一对。TIMP-1可与MMP-9前体(pro-MMP-9)形成稳定的复合体,阻碍pro-MMP-9的自我激活；同时TIMP-1抑制已活化的MMP-9的活性,直接与活化的MMP-9 Zn2+活性中心按1：1比例发生不可逆结合,阻断或抑制MMP-9活性,而MMP-9活性高低直接影响肾间质纤维化的进程。在不同病因所致的肾间质纤维化病程发展中均存在MMPs/TIMPs的功能紊乱。不同实验方法观察可有不同的结果,但有一种共同趋势,即TIMPs高表达,和(或)MMPs的活性下降。寻找MMPs激活剂或表达促进剂或者TIMPs的抑制剂,有可能成为预防和治疗肾间质纤维化的新方法。
中性粒细胞明胶酶相关脂质运载蛋白,英文名称为Neutrophil gelatinase-associated lipocalin(NGAL),又称为lipocalin-2,是lipocalin家族新成员,是1993年K Jeldsen在中性粒细胞中发现的一种小分子量分泌性蛋白。NGAL除具有lipocalin家族共有疏水基结构外,尚存在一游离的巯基,可与MMP-9前体等其他分子结合,为其发挥特殊的生物学作用奠定了基础。NGAL在人体表达于多种组织器官,如：支气管、胃、小肠、肾脏、前列腺、胸腺等。NGAL具有参与炎症趋化与免疫应答反应；参与肾小管上皮细胞的形成、修复；参与肿瘤的发生发展等多种生物学作用。
研究发现NGAL对调节MMP-9/TIMP-1具有一定作用。NGAL可结合于MMP-9上保护MMP-9不被降解。一般情况下,MMP-9是以Pro-MMP-9形式贮存于细胞内,分泌到细胞外之后,只有切去N端的一段序列,MMP-9才表现活性。通常,Pro-MMP-9在细胞内主要以四种方式存在：单体、同源二聚体、NGAL与Pro-MMP-9以分子间二硫键结合形成的异源二聚体(NGAL/Pro-MMP-9)以及此异源二聚体与金属蛋白酶组织抑制剂-1(TIMP-1)形成的三元复合物NGAL/ Pro-MMP-9/TIMP-1。在这种三元复合物中,Pro-MMP-9仍可被激活为有活性的MMP-9,但活性会有所降低。然而当此三元复合物中的TIMP-1通过它的游离N-端结构域去结合和抑制其它的MMPs时,又可形成NGAL/Pro-MMP-9/ TIMP-1/MMPs四元复合物。而当这种四元复合物被活化时,Pro-MMP-9被激活成MMP-9的活性反而比三元复合物时的活性高许多倍。很显然NGAL明显具有调节这些复合物的能力,甚至从某种程度上来讲,NGAL具有抵消复合物中TIMP-1抑制MMP-9活性的作用。故通过探讨NGAL在肾间质纤维化模型中表达情况及与,对于探索其在肾间质纤维化中的作用机制及为治疗肾间质纤维化可能提供新思路。
研究发现急性药物相关性肾炎患者肾组织中NGAL在近端小管的表达较正常人增强,NGAL表达量与间质-小管损害呈负相关,说明NGAL在急性肾小管间质损伤中起到一定保护作用,但具体机制尚不明确,推测NGAL可能在肾间质纤维化中发挥一定作用。Jaya Mishra在急性小鼠急性缺血-再灌注损伤模型中发现,予NGAL治疗可显著减少小管细胞组织病理学损伤,增加近端小管数目,推测NGAL可能诱导肾间质细胞向小管上皮细胞转换而诱导肾小管上皮细胞再生。
目前国内外对NGAL研究多集中于其在乳腺癌等肿瘤发生机制中的作用及在肾脏急性缺血-再灌注损伤、肾移植及心肺分流术等多种因素诱发的急性肾功能损害的早期检测指标等。对于NGAL在肾间质纤维化中的作用研究较少,以上研究均证明NGAL在肾间质纤维化过程中可能起到一定的调节作用,故探讨NGAL在肾间质纤维化模型中表达情况及作用,对于探索其在肾间质纤维化中的作用机制及为治疗肾间质纤维化可能提供新思路。
研究目的
1、动态观测单侧输尿管梗阻大鼠梗阻侧肾组织NGAL、MMP-9及TIMP-1的表达变化,探讨NGAL与MMP-9、TIMP-1在肾纤维化发展过程中的相关关系;
2、探讨NGAL在肾小管间质纤维化发展中的作用机制。
研究方法
1、制作单侧输尿管梗阻模型
将48只雄性SD大鼠随机分为单侧输尿管梗阻组(UUO)及假手术组(SOR),UUO组行左侧输尿管结扎术,SOR组仅游离左侧输尿管不结扎。术后第1、4、7、14天分别处死每组中的6只大鼠,取左肾(梗阻侧肾脏)行HE和Masson染色。
2、用免疫组化ELiVisionTMplus法检测肾组织NGAL、MMP-9、TIMP-1及FN的表达情况。
3、用SPSS13.0对结果进行数据分析。
结果
1、HE、MASSON染色评估肾纤维化程度
UUO组第1天远端小管无扩张,肾间质无明显水肿；术后第4天远端小管扩张,肾间质轻度水肿,间质内散在淋巴细胞、巨噬细胞浸润；术后第7天远端小管扩张明显,肾间质明显水肿,近端小管出现扩张,炎性细胞浸润进行性加重并伴有纤维细胞增生；术后第14天可见肾小管、集合管扩张呈囊状,部分上皮细胞变性、坏死,肾小管结构破坏,管腔塌陷,炎性细胞浸润。
2、FN免疫组化结果
SOR组大鼠FN在肾小球、肾小管基底膜及肾间质有少许表达,UUO组随
Reserch background
The reserch shows that there is close relation between renal interstitial disease and the progression of chronic kidney failure. Renal interstitial fibrosis is a common feature of progressive renal diseases. It is a very hot pot to study the pathological process and mechanism of renal interstitial disease and search a new target for delaying and blocking renal the progression of renal fibrosis. A great many cytokines and biotic activators participate the genesis and development of renal interstitial fibrosis. All regulatory factors were connected through the biological signal-transducting mechanism, forming the consummate regulatory feedback system to regulate the pathological and physiological processes of organism.
The generation and development of renal interstitial fibrosis are complicated. The main course include the fibroblast's activation, generation and phenotype transition, making it transform into myofibroblast, which can secrete a-smooth muscel actin. So that the generation of extracellular matrix(ECM) increaes, and the balance of ECM's production and degradation collapse with the regulation of mutiple cytokines and inflammatory cell infiltration etc, resulting in the accumulation of ECM and the progression of renal interstitial fibrosis. There are multiple cytokines participating the pathological process, including the promoting factors such as transforming growth factor beta-1(TGF-β1), connective tissue growth factor(CTGF), platelet-derived growth factor(PDGF) and the inhibiting factors like hepatocyte growth factor(HGF), decrin, and bone morphogenetic protein-7(BMP-7) etc. TGF-β1 is considered as the most important promoting fibrosis factor of the mutiple promoting cytokines, and most researchers tried to block the progression of Renal interstitial fibrosis by restrainning the TGF-β1. But 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 death. So it's important to find new targets for supressing the development of RIF without hurting the physiologic function of organism.
Matrix metalloproteinases(MMPs) are very important enzymes degrding ECM. MMPs'functions include ECM degration and activating other MMPs causing cascade effects. MMPs were secreted into ECM in form of proenzyme and turn into bioactive MMPs educing biological activity by taking off propeptide with reactivator's effect. Tissue inhibitors of metalloproteinase(TIMPs) can restrain proenzyme and enzyme activity of MMPs and play important roles in regulating the production and degradation of ECM. Renal interstitial fibrosis is mainly caused by accumulation interstitial collagen of typeⅠ、Ⅱ、ⅢandⅣ, glucoprotein and proteoglycan. Imbalance of MMPs and TIMPs is the key point of progression of renal interstitial fibrosis, while MMP-9 and TIMP-1 is the most important pair. TIMP-1 can combine MMP-9 precurosor turning into stable complex and hinder its self-activation. Meanwehile TIMP-1 can retrain activated MMP-9's activity by irreversibly combining its active center with Zn2+by ratio of one by one. However the activity of MMP-9 directly influences the progression of renal intersitial fibrosis. There are functional disorders of MMPs/TIMPs in the progression of renal interstitial fibrosis caused by various origins of diseases. There might be different outcomes using different methods, but there is a common tendency which is the hypo-expression of the TIMPs with or without the lower activity of MMPs. Therefore it is demanded to look for better activators for MMPs or restrainers for TIMPs which might be a new method to prevent and cure renal fibrosis.
Neutrophil gelatinase-associated lipocalin, short for NGAL, also called lipocalin 2, is a new member of the lipocalin family, which was found as a scecreted protein from neutrophilic leukocyte by K jeldsen in 1993. Besides the common structure of hydrophobic group that the lipocalin family have, it still has the nomadic hydrosulfide group, which can combine MMP-9 precurosor and other molecules, establishing foundation for its special biological contribution. NGAL protein expressed in many organs in human body, such as bronchus, stomach, small intestinum, kidney, prostate and thymas gland. NGAL protein can educe its multiple biological effects by participating immune response reaction, the formation and reparation of renal tubular epithelial cells, and generation and progression of tumors.
It is discovered that NGAL protein plays roles in regulating MMP-9 and TIMP-1.NGAL can combine MMP-9 protecting it from degradation. MMP-9 protein is ususlly stored in cells in form of MMP-9 precurosor, and activated after the series of N-apical being cut when secreted into extracelluar enviroment. MMP-9 precurosor usually exsit in four forms:monomer, homodimer, heterodimer of NGAL protein and MMP-9 precurosor connected by intermolecular disulfide bond, and the ternary complex combined with TIMP-1 by heterodimer. MMP-9 precurosor could still be activated in the ternary complex but get lower activity compared with the monomer type. However TIMP-1 of the ternary complex can combine other MMPs protein by its nomadic N apical structural domain, turning into quaternary complex. When MMP-9 precurosor of the quaternary complex is activated, it has much more activity than that of the ternary one. Thus NGAL protein apparently has the ability of regulating the complex mentioned above, which can counteract the supression of TIMP-1 on MMP-9's activity. It might be useful in obseving NGAL's expression in the model of renal interstitial fibrosis to investigate its mechanism of action in RIF and look for new ideas of curing RIF.
Researchers found that the expression of NGAL protein strenthened in proximal tubules in the patients with acute drug induced interstitial nephritis compared with that of normal persons. The expression was negative correlated with the damage of interstitium, which meant that NGAL educed protective effects in the actue interstitial damage, but the spicific mechanism hadn't been identified, speculating that NGAL might be effective in the RIF progression. Jaya Mishra found that NGAL therapy could decrease the pathological damage in tubular epithelial cells significantly and the proximal tubules increased by creating the acute ischemic-reperfusion injury model of mouse, speculating that NGAL might induce interstial cells transforming into epithelial cells, contributing to the rebirth of renal epithelial cells.
The research of NGAL protein at present mostly focus on its effect on the mechanism of tumors such as breast cancer and etc, and as an early detecting cytokine in the acute renal failure caused by renal ischamic-reperfusion injury, kidney transplantation, cardiopulmonary bypass and etc. There are few reaseaches on its effects in renal interstitial fibrosis, however the investigations mentioned above proved that NGAL might play roles in the progression in RIF. So it's necessary to investigate its expression in RIF model and find new methods to prevent and cure RIF.
Objective
1. To observe the expression of NGAL, MMP-9 and TIMP-1 in renal tissues of rats following unilateral uretral obstuction(UUO), and investigate the correlation of cytokines above in the progression of renal interstitial fibrosis.
2. To investigate the mechanism of action for NGAL in the progression of RIF.
Methods
1. Forty-eight male Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group and an unilateral uretral obstruction group. UUO model was induced by ligating the left ureter of rats. Six rats in each group were sacrificed on the 1st,4th,7th and 14th day after UUO.
2、Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of NGAL, MMP-9, TIMP-1 and FN were detected by immunohistochemical staining of ELiVisionTMplus.
3、Analysis of data with SPSS 13.0 software.
Results
1. On the 1st day, there was little expasion in the distal tubules and little edema in the interstitium. On the 4th day there was renal tubular ectasia, scattered leukocyte and macrophage, interstitial cells were partialy denaturated in the UUO group. When the 7th day came, there was severe edema in interstitium and more infiltration of inflammatory cells and proliferation of fibrocyte. necrosis of interstitial cells in renal tubule and an increase of fiber in interstitial substance. Till the 14th day, there was asystematic thickening and shrinkage of renal tubular basement membrane, and the fibrosis was obvious. 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 for FN indicated that:there was only a little expression of FN in renal glomeruli, tubular basement membrane and renal interstitium. While in the UUO group the expression of FN became higher and higher as obstructed time went(P<0.05). And the positive degree was much higher in UUO group than that of SOR group at each time point respectively(P<0.05).
3. NGAL expressed in the proximal tubules in renal tissue in SOR group. Its expression expanded and strengthened in the early stage of RIF(on the 1st and 4th day after UUO), however it decreased as time went(on the 7th and 14th day), still higher than that of SOR group(P<0.05).
4. MMP-9 expressed in the proximal tubular epithelial cells in renal tissue in SOR group. The expression of MMP-9 strengthened in the UUO group, rising in the early stage(from 1st to 7th day) and decreasing after 7th day(P<0.05).
5. There was little expression of TIMP-1 in SOR group. After obstruction the expression of TIMP-1 gradually increased as time went, and the positive degree was higher in the UUO group than that of SOR group(P<0.05).
Conclusion
The expression of NGAL increased on the early stage after obstruction indicated that it played roles in the early stage of renal interstitial fibrosis. There was apparent. negative correlation between NGAL positive degree and the injury index of interstitium and fibrosis degree meaning that it might restrain the progression of RIF. There was positive correlation between NGAL and the ratio of MMP-9 and TIMP-1 indicating that NGAL delayed the progression of RIF by regulating the balance of MMP-9/TIMP-1.
引文
[1]Vu T H, Werb Z. Matrix metalloproteinases:effectors of development and normal physiology.[J]. Genes Dev.2000,14(17):2123-2133.
[2]Chevalier R L. Pathogenesis of renal injury in obstructive uropathy.[J]. Curr Opin Pediatr.2006,18(2):153-160.
[3]Liu Y, Rajur K, Tolbert E, et al. Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. [J]. Kidney Int.2000,58(5):2028-2043.
[4]Uchio-Yamada K, Manabe N, Goto Y, et al. Decreased expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in the kidneys of hereditary nephrotic (ICGN) mice.[J]. J Vet Med Sci.2005,67(1):35-41.
[5]Bode W, Fernandez-Catalan C, Tschesche H, et al. Structural properties of matrix metalloproteinases.[J]. Cell Mol Life Sci.1999,55(4):639-652.
[6]Kjeldsen L, Johnsen A H, Sengelov H, et al. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase.[J]. J Biol Chem. 1993,268(14):10425-10432.
[7]Coles M, Diercks T, Muehlenweg B, et al. The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin.[J]. J Mol Biol.1999,289(1): 139-157.
[8]Bratt T, Ohlson S, Borregaard N. Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands.[J]. Biochim Biophys Acta.1999,1472(1-2):262-269.
[9]Yan L, Borregaard N, Kjeldsen L, et al. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.[J]. J Biol Chem.2001,276(40):37258-37265.
[10]Kolkenbrock H, Hecker-Kia A, Orgel D, et al. Progelatinase B forms from human neutrophils. complex formation of monomer/lipocalin with TIMP-1.[J]. Biol Chem.1996,377(7-8):529-533.
[11]刘克剑,张悦,李靖,何立群.单侧输尿管梗阻法制作大鼠肾间质纤维化模型的改进[Z].2007：15,410-412.
[12]Eitner F, Floege J. Novel insights into renal fibrosis.[J]. Curr Opin Nephrol Hypertens.2003,12(3):227-232.
[13]Kalluri R, Neilson E G. Epithelial-mesenchymal transition and its implications for fibrosis.[J]. J Clin Invest.2003,112(12):1776-1784.
[14]Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis.[J]. J Mol Med.2004,82(3):175-181.
[15]Yang J, Dai C, Liu Y. A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.[J]. J Am Soc Nephrol.2005, 16(1):68-78.
[16]Rastaldi M P. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis.[J]. J Nephrol.2006,19(4):407-412.
[17]Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis:pathologic significance, molecular mechanism, and therapeutic intervention.[J]. J Am Soc Nephrol.2004,15(1):1-12.
[18]Vongwiwatana A, Tasanarong A, Rayner D C, et al. Epithelial to mesenchymal transition during late deterioration of human kidney transplants:the role of tubular cells in fibrogenesis.[J]. Am J Transplant.2005,5(6):1367-1374.
[19]Yang J, Shultz R W, Mars W M, et al. Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy.[J]. J Clin Invest.2002,110(10):1525-1538.
[20]Lan H Y. Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells.[J]. Curr Opin Nephrol Hypertens.2003,12(1):25-29.
[21]李德天杨.骨形态发生蛋白7在单侧输尿管梗阻大鼠肾组织中的表达[Z].2007：36,535-538.
[22]Yang J, Goetz D, Li J Y, et al. An iron delivery pathway mediated by a lipocalin.[J]. Mol Cell.2002,10(5):1045-1056.
[23]Mori K, Lee H T, Rapoport D, et al. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.[J]. J Clin Invest.2005,115(3):610-621.
[24]Kim B S, Yoon K H, Oh H M, et al. Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death.[J]. Cell Immunol.2002,220(2):96-106.
[25]Dai C, Yang J, Liu Y. Transforming growth factor-betal potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling.[J]. J Biol Chem.2003,278(14):12537-12545.
[26]Hanai J, Mammoto T, Seth P, et al. Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.[J]. J Biol Chem.2005,280(14):13641-13647.
[27]Benyon R C, Arthur M J. Extracellular matrix degradation and the role of hepatic stellate cells.[J]. Semin Liver Dis.2001,21(3):373-384.
[2]Chevalier R L. Pathogenesis of renal injury in obstructive uropathy.[J]. Curr Opin Pediatr.2006,18(2):153-160.
[3]Liu Y, Rajur K, Tolbert E, et al. Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. [J]. Kidney Int.2000,58(5):2028-2043.
[4]Uchio-Yamada K, Manabe N, Goto Y, et al. Decreased expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in the kidneys of hereditary nephrotic (ICGN) mice.[J]. J Vet Med Sci.2005,67(1):35-41.
[5]Bode W, Fernandez-Catalan C, Tschesche H, et al. Structural properties of matrix metalloproteinases.[J]. Cell Mol Life Sci.1999,55(4):639-652.
[6]Kjeldsen L, Johnsen A H, Sengelov H, et al. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase.[J]. J Biol Chem. 1993,268(14):10425-10432.
[7]Coles M, Diercks T, Muehlenweg B, et al. The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin.[J]. J Mol Biol.1999,289(1): 139-157.
[8]Bratt T, Ohlson S, Borregaard N. Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands.[J]. Biochim Biophys Acta.1999,1472(1-2):262-269.
[9]Yan L, Borregaard N, Kjeldsen L, et al. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.[J]. J Biol Chem.2001,276(40):37258-37265.
[10]Kolkenbrock H, Hecker-Kia A, Orgel D, et al. Progelatinase B forms from human neutrophils. complex formation of monomer/lipocalin with TIMP-1.[J]. Biol Chem.1996,377(7-8):529-533.
[11]刘克剑,张悦,李靖,何立群.单侧输尿管梗阻法制作大鼠肾间质纤维化模型的改进[Z].2007：15,410-412.
[12]Eitner F, Floege J. Novel insights into renal fibrosis.[J]. Curr Opin Nephrol Hypertens.2003,12(3):227-232.
[13]Kalluri R, Neilson E G. Epithelial-mesenchymal transition and its implications for fibrosis.[J]. J Clin Invest.2003,112(12):1776-1784.
[14]Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis.[J]. J Mol Med.2004,82(3):175-181.
[15]Yang J, Dai C, Liu Y. A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.[J]. J Am Soc Nephrol.2005, 16(1):68-78.
[16]Rastaldi M P. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis.[J]. J Nephrol.2006,19(4):407-412.
[17]Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis:pathologic significance, molecular mechanism, and therapeutic intervention.[J]. J Am Soc Nephrol.2004,15(1):1-12.
[18]Vongwiwatana A, Tasanarong A, Rayner D C, et al. Epithelial to mesenchymal transition during late deterioration of human kidney transplants:the role of tubular cells in fibrogenesis.[J]. Am J Transplant.2005,5(6):1367-1374.
[19]Yang J, Shultz R W, Mars W M, et al. Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy.[J]. J Clin Invest.2002,110(10):1525-1538.
[20]Lan H Y. Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells.[J]. Curr Opin Nephrol Hypertens.2003,12(1):25-29.
[21]李德天杨.骨形态发生蛋白7在单侧输尿管梗阻大鼠肾组织中的表达[Z].2007：36,535-538.
[22]Yang J, Goetz D, Li J Y, et al. An iron delivery pathway mediated by a lipocalin.[J]. Mol Cell.2002,10(5):1045-1056.
[23]Mori K, Lee H T, Rapoport D, et al. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.[J]. J Clin Invest.2005,115(3):610-621.
[24]Kim B S, Yoon K H, Oh H M, et al. Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death.[J]. Cell Immunol.2002,220(2):96-106.
[25]Dai C, Yang J, Liu Y. Transforming growth factor-betal potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling.[J]. J Biol Chem.2003,278(14):12537-12545.
[26]Hanai J, Mammoto T, Seth P, et al. Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.[J]. J Biol Chem.2005,280(14):13641-13647.
[27]Benyon R C, Arthur M J. Extracellular matrix degradation and the role of hepatic stellate cells.[J]. Semin Liver Dis.2001,21(3):373-384.