肾淋巴循环障碍对肾脏结构及功能影响的实验研究
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摘要
背景和目的:
随着人民生活水平的提高及寿命的延长,慢性肾脏病(CKD)的发病率明显增高。慢性肾损害所致的尿毒症给病人带来了极大的痛苦及经济负担。因此探讨慢性肾损害的原发致病因素并采取早期干预措施有重大理论意义和临床实用价值。近年来,有关器官内淋巴管的超微结构及其循环障碍对组织影响的研究已有一些报道,尤其在脑、肝脏、胃肠等器官已取得了较大进展。然而,关于肾脏毛细淋巴管的超微结构以及循环障碍对肾脏影响的研究少见报道。早期曾有人研究过短时结扎犬肾门淋巴管后,对肾功能的影响,发现肾间质静水压增高,钠水排泄改变,尿量增加,随着时间延长,可出现肾间质的弥漫性损害。那么,肾脏淋巴循环和肾脏组织结构改变之间有着怎样的关系,又是通过何种机制起作用呢?至今尚无人深入研究。
我们早期的预实验发现结扎大鼠肾淋巴管后,尿蛋白显著增加,随着时间推移肾功能逐渐减退,组织病理出现以肾小管间质损伤为主的改变,包括上皮细胞崩溃脱落坏死,小管萎缩,正常结构破坏,肾间质纤维化,肾小球系膜细胞及基质增生等。我们推测肾淋巴循环障碍将激发肾脏纤维化与肾脏细胞凋亡的发生。我们知道肾间质纤维化是所有慢性肾脏疾病的共同特征,几乎是所有终末期肾病(ESRD)的最终转归,而肾小管上皮细胞-间充质细胞转化(EMT)是肾间质纤维化的重要发病机制之一。上皮细胞-间充质细胞转化过程的经典调控因子为转化生长因子-β超家族成员。SMAD家族蛋白的主要作用是介导TGF-β家族的信号。细胞凋亡又称程序性的细胞死亡,随着对细胞凋亡研究不断深入,发现Bcl-2家族是在细胞凋亡中有重要作用的一类蛋白质。Bcl-2家族的表达和调控是影响细胞凋亡的关键因素之一,在细胞凋亡信号转导途径中发挥重要作用。Bcl-2和bax分别是bcl-2家族中最有代表性的抑制凋亡和促进凋亡基因,并且bax是bcl-2活性的主要调控因子。尽管,对肾间质纤维化和细胞凋亡的研究已较深入广泛,但肾淋巴循环障碍是否引起肾间质纤维化和细胞凋亡,TGF-β_1/smad途径和bax/bcl-2途径在其中的作用如何却仍不清楚。
本课题吸收国内外研究的最新成果,结合国内外肾脏病防治的迫切需要,通过动物试验,用结扎肾淋巴管的方法建立肾淋巴循环障碍大鼠模型,通过观察肾组织病理及功能变化用以证实肾淋巴循环障碍所导致的肾脏损害,通过对TGF-β/SMAD途径和Bcl-2/bax途径的相关分子蛋白的检测,阐明其肾间质纤维化及肾细胞凋亡的发生机制,为临床越来越多的不明原因的慢性肾损伤寻找新的原发致病因素。
研究方法:
1.鼠肾起始淋巴管的分布及形态学观察
1.1 Wistar大鼠20只,雌雄不限。常规麻醉、手术开腹。每只动物取肾组织用免疫荧光双标法,即淋巴管标记物Podoplanin和血管内皮标记物CD34分别标记淋巴管和血管,应用Leica荧光显微镜在不同的通道(绿光激发光波长543nm;红色激发光波长633nm)分别观察并拍照,以观察鼠肾起始淋巴管在肾脏分布。
1.2肾脏超微结构观察:在皮质,髓质及皮髓交界区取组织块置3%戊二醛固定液内,用透射电镜观察淋巴管超微结构,包括毛细淋巴管的一般特征、内皮细胞的连接、质膜小泡的形态和分布。
2.对大鼠肾脏结构功能的观察
2.1将大鼠分为两组:模型组和对照组,每组30只。用结扎肾淋巴管的方法建立了肾淋巴循环障碍大鼠模型。各组大鼠分别于术后第7、14、28、56天各处死6只。在处死前1日留取24小时尿标本,处死日留取血标本。尿蛋白和血清肌酐由全自动分析仪检测。Ccr按公式:尿肌酐浓度×每分钟尿量(ml)/血肌酐浓度计算,并以体重校正。以观察不同时间段大鼠尿蛋白、肾功能改变。
2.2肾脏组织经固定、脱水、石蜡包埋,切片3μm厚度,做PAS、Masson染色。在光镜下观察肾脏病理改变,并根据小管损伤,小管间质纤维化和肾小球硬化标准作小管损伤指数,小管间质纤维化指数(TIFI)和肾小球硬化指数(GSI)的半定量分析。
2.3留取约1mm~3大小肾皮质经固定、包埋,切片厚50nm,观察肾脏超微结构改变。
3.淋巴循环障碍促使肾间质纤维化的研究
假手术组和模型组大鼠分别于术后第7、14、28、56天处死。留取肾组织标本提取组织蛋白、mRNA基因和制作石蜡切片。运用Real-time PCR、Western Blot和免疫组化检测检测TGF-β_1、Smad2/3、P-smad2/3在肾脏组织的表达。
4.肾淋巴循环障碍促使肾细胞凋亡的研究
各组大鼠分别于术后第1、7、14、28天各处死6只,留取肾组织标本提取组织蛋白、mRNA基因和制作石蜡切片。运用Real-time PCR、Western Blot和免疫组化检测检测Bax、Bcl-2、Caspase3在肾脏组织的表达,用肾脏细胞原位凋亡(TUNEL)的方法检测肾脏凋亡细胞。
结果:
1.鼠肾起始淋巴管的分布及形态学观察
1.1经免疫荧光双重染色,光镜下可见毛细血管呈红色、毛细淋巴管呈绿色,区别明显。淋巴管形态不规则,管腔大,壁薄。淋巴管主要位于富含结缔组织区,大鼠肾脏皮质淋巴管明显多于髓质。
1.2电镜下见毛细淋巴管的形状极不规则,毛细淋巴管的管壁较薄,毛细淋巴管内皮细胞无窗孔,基膜很薄,不连续或缺如,可见细胞内通道样结构,淋巴管内皮细胞中富含细胞器和质膜小泡。内皮细胞以重叠连接为主,在内皮细胞的连接处可见有粘着装置。毛细淋巴管的内皮细胞中含有极其丰富、大小不等的质膜小泡。质膜小泡多数游离于细胞质内。
2.对大鼠肾脏结构功能的观察
2.1模型组大鼠于术后第1周即出现明显蛋白尿(P＜0.05),随时间的延长而加重;模型组大鼠血肌酐水平在第2周时开始升高,(P＜0.05),且随术后时间延长,有逐渐增高趋势;模型组Ccr水平从术后第2周起明显下降,以后随着术后时间的延长,不断下降。
2.2模型组大鼠出现明显的组织病理改变,肾小管扩张明显,伴上皮细胞空泡变性,部分小管出现上皮细胞崩溃脱落,小管结构不完整。随术后时间延长出现肾小管萎缩,间质面积增宽,小管和间质结构消失,其中可见大量纤维组织增生。肾小球系膜区可见系膜细胞及基质增生。小管损伤指数,小管间质纤维化指数(TIFI)和肾小球硬化指数(GSI)较对照组明显增高,并随病程逐渐进展。
2.3电镜下观察模型组于造模后1周即出现肾小管上皮细胞、肾小球足细胞水肿,胞质内空泡数目增加,线粒体肿胀,部分嵴消失部分线粒体嵴消失呈空泡状;足突融合、变平;系膜增生插入,基质呈高电子密度。
3.淋巴循环障碍促使肾间质纤维化的研究
3.1免疫组化显示,模型组大鼠在小管上皮及系膜区的TGF-β_1、Smad2/3表达明显增强,在小管上皮细胞更为明显。ColⅠ在肾间质及肾小管上皮细胞的表达明显增高,而肾小球无明显变化。p-Smad2/3主要表达于肾小管上皮细胞,在肾小球系膜细胞的细胞核内也可见较强的p-Smad2/3的表达。
3.2 Western印迹检测发现模型组TGF-β_1、Smad2/3的蛋白表达量较对照组明显增加(P＜0.05),在第1周开始就有p-Smad2/3蛋白表达,第2周后持续增高,以后维持在较高水平(P＜0.05)。
3.3 Real-time PCR的检测显示ColⅠ、TGF-β_1、Smad2、Smad 3的mRNA均于术后第1周开始增高,分别为同期对照组的4.5、1.6、1.7、1.8倍(P＜0.05)。
4.肾淋巴循环障碍促使肾细胞凋亡的研究
4.1应用TUNEL检测发现,在模型组大鼠阳性着色细胞显著增加,且细胞凋亡主要发生在扩张或萎缩的肾小管;模型组的凋亡指数在术后第一天开始升高(p＜0.05),在第14天达到高峰(p＜0.01)。
4.2免疫组化提示,模型组的Bax和caspase-3表达明显增强,主要位于小管间质,尤其在远端小管。相反,Bcl-2的表达明显减弱。
4.3 Western blot分析显示,模型组大鼠Bax蛋白表达水平明显增加,在术后第14天达到高峰(p＜0.01),以后维持缓慢增高趋势。然而Bcl-2蛋白表达从术后第一天开始明显下降。由此,Bax/Bcl-2比值明显增高。总caspase-3表达明显高于对照组(p＜0.01),且其活化片段(17-kDa)表达也明显增高。
4.4 Real-time PCR显示在所有时间段,模型组大鼠bax和caspase-3的mRNA表达显著增高,而bcl-2 mRNA表达却明显下降(p＜0.05)。同样Bax/Bcl-2比值也较对照组明显增高。
结论:
1.大鼠肾脏淋巴系统内大量质膜小泡和通道样结构的存在,正是肾脏转运大分子物质和组织液进入淋巴循环的生理基础。
2.肾淋巴循环障碍对肾脏结构和功能有着明显的影响,肾脏功能减退和结构损害随循环障碍时间的延长而加重。
3.在肾淋巴循环障碍导致的小管间质纤维化过程中,TGF—β_1/Smad途径被高度激活,促进着上皮细胞的转型,在纤维化过程中起着积极作用。
4.在肾淋巴循环诱导的肾脏功能和结构改变的过程中,细胞凋亡显著增加,其中Bax/Bcl-2途径发挥了积极作用。
Background and objective:
Recent surveys have revealed that the prevalence of chronic kidney disease (CKD) is surprisingly high in the general population. Most patients with CKD develop end-stage renal failure, which requires costly treatment and unaffordable renal replacement therapy. Therefore, to find out the cause of chronic renal damage and to develop the effective treatment seemed to be emergency. Renent year, the study of lymph ultrastructure in some organs and their circlulation disorders have been done, especially in brian, liver, and stomach intestine. However, there is very few about renal lymph ultrastructure and the effect of its disorder on renal function and structure. The knowledge on renal lymphatics is very limited and their importance is almost always neglected. The role of lymph in keeping the balance between reabsorption of fluid from the tubules and uptake of reabsorbed fluid by the peritubular capillaries is highlighted. Early papers demonstrated that acute ligation of hilar lymphatic ducts has a significant effect on renal function, such as changes of sodium excretion, urine volume. As long as the time goes by, it appears diffuse lesion in renal interstitium. Therefore, what is the relationship between renal lymph circlulation and renal structure, what is the exact mechanism to interfere with? There is nobody to study.
In our pre-study, we demonstrated that rats developed significant and progressive proteinuria and elevating serum creatinine, when renal hilar lymph ducts was ligated. These kidneys were characterized by tubular cell detachment, tubular cell necrosis, tubular atrophy or dilatation and deposition of ECM. Thus,we suggest that renal lymph disorder induces renal fibrosis and renal cell apoptosis. As we know, tubulointerstitial fibrosis is critical determinants of renal function, and is the common final way of end stage of renal disease. The renal epithelial -mesenchymal transition(EMT) is one of the important mechanism of tubulointerstitial fibrosis. It is well known that TGF-β1 plays a significant role in the progression of EMT. Smad proteins have been identified recently as important components of the TGF-β_1 signaling pathway. Apoptosis is so called programmed cell death, and rencent study revealed that Bcl-2 family members are significant to cell apoptosis. The expression and regulation of Bcl-2 family proteins play a key role in apoptotic signal transduction pathway. Proapoptotic Bax and antiapoptotic Bcl-2 are the typical genes in Bcl-2 family, and furthermore, bax is the chief active regulating factor of bcl-2. In this study, we established the rat model of renal lymph circlulation disorder to show the time course of renal lesions caused by lymphatic ligation ; and to reveal the exact mechanism of renal lesion through detect the molecule involved in TGF-β_1/ SMAD and Bcl-2 /bax pathway.
Methods:
1. The study of the distribution and morphologic feature of the intrarenal lymphatics
1.1 There are 20 female and male Wistar rats were involved in this study and nephridial tissue were obtained from each animal. Double immunofluorescence was applied to detect the lymphatic distribution by using Podoplanin and CD34. Using Leica fluorescence microscope , green light was detect under 543nm and red light under 633nm wavelength of light.
1.2 Observe of renal lymphatic ultrastructure :renal tissues were obtained from the cortex, medulla and junctional zone , and put in 3% glutaric dialdehyde stationary liquid for transmission electron microscope to observe the fine distribution and morphologic feature of the intrarenal lymphatics.
2. The study of rats renal function and structure
2.1 Animals were divided into two groups: control group and model group (30 rats in each group) .Establish the rat model of renal lymph circlulation disorder by renal lymph ligation. Six rats in each group were sacrifice at 7、14、28、56d after operation. Urine aliquot and blood preparation were obtained. Proteinuria and serum creatinine value were examed by automatic analyser .Creatinine clearance (Ccr) formula : urine creatinine×urine volume per minute/serum creatinine, corrected by body weight.
2.2 The renal tissue was examined by PAS and Masson staining after fixation, dehydration, paraffin imbedding. The assessment of tubulus injury index , TIFI and GSI was performed with the use of semiquantitative scores .
2. 3 Obtained 1mm~3 of renal cortex for fixation, embedding sectioning. The ultrastructure of renal tissue was detected by electron microscope.
3. The study of renal interstitial fibrosis induced by renal lymph circulation disorder
Rats in each group were sacrifice at 7、14、28、56d after operation. Nephridial tissue were obtained for extracting protein , mRNA and paraffin section. Real-time PCR, immunostaining and western blot techniques were applied to detect the expression of COLI , TGF-β_1, Smad2/3, P-smad2/3 in renal tissue.
4. The study of renal cell apoptosis induced by renal lymph circlulation disorder
Rats in each group were sacrifice at 7、14、28、56d after operation. Nephridial tissue were obtained for extracting protein , mRNA and paraffin section. Real-time PCR, immunostaining and western blot techniques were applied to detect the expression of Bax/Bcl-2, Caspase3 in renal tissue. Apoptotic nuclei were labeled using the terminal deoxynucleotidyltransferase dUTP nick-end-label (TUNEL) technique.
Results:
1. The study of the distribution and morphologic feature of the intrarenal lymphatics
1.1 Under immunofluorescence double staining, blood capillary showed schillerization, and lymphatic capillary showed green color. lymphatic capillary appeared anomalism, large lumina , thin wall and located in connective tissue region. Cortilymph obviously exceed medulla lymph in rats.
1.2 Under electron microscope, the basal lamina of lymphatic was discontinuity.A lot of lysosome and vesicles existed in the endothelial of capillary lymphatics. Many of the vesicles liberated in cytoplasm. The intercellular junctions of the renal lymphatics are held in close apposition.
2. The study of rats renal function and structure
2.1 Model group rats developed severe proteinuria and elevated serum creatinine and reduced creatinine clearance were observed.
2.2 Histomorphological changes appeared in ligated kidneys, characterized by tubular damage, tubulointerstitial fibrosis, and expansion of the mesangium. The index of tubular damage and tubulointerstitial fibrosis were remarkably aggravated in model group.
2.3 Under electron microscope, renal tubular epithelial cell andpodocyte showed edema; the number of vacuolus increased in intracytoplasm; mitochondrial crista disappeared; foot process got fusion; mesenterium got proliferation and insertion;ground substance became electron-dense
3. The study of renal interstitial fibrosis induced by renal lymph circlulation disorder
3.1 Immunohistochemical staining indicated that Overexpression of transforming growth factor-β_1 (TGF-β_1) , Smad2/3, COL I genes and proteins were detected in model group rats. These proteins were mainly expressed in renal tubulointerstitium .
3.2 Western blot showed protein expression of TGF-β_1 , Smad2/3 were increased in model group rats. The expression of p-Smad2/3 increased from week 1 and keep high level after then.
3.3 Real-time PCR revealed that the expression of Col I, TGF-β_1, Smad2, Smad 3 mRNA were up-regulation from week 1, as 4.5、1.6、1.7、1.8 folds as control group.
4. The study of renal cell apoptosis induced by renal lymph circlulation disorder
4.1 TUNEL exam showed that many positive staining cells in model group rats. cell apoptosis mainly occured in dilate and emarcide renal tubule. Apoptotic index elevated from day 1 in model group , peaked at day 14.
4.2 Immunohistochemical staining indicated that overexpression of Bax and caspase-3 in model group rats, mainly in renal tubulointerstitium. Oppositely, Bcl-2 was weakly.
4.3 Western blot showed protein expression of Bax, caspase-3 increased in model group rats, peaked at day 14. The active unit of caspase-3 (17 KD) was also elevated in model group. Icreasing bax to bcl-2 ratio were also detected in model group rats.
4. 4 Real-time PCR show that the expression of bax and caspase-3 were up-regulation in all time points. However, bcl-2 mRNA decreased in model group. Icreasing bax to bcl-2 ratio were also detected in model group rats. Conclusions:
1. The intercellular junctions of the renal lymphatics are held in close apposition by adhesion devices and a lot of lysosome and vesicles existed in the endothelial of capillary lymphatics, which are the base for renal to transport big molecular and fluids.
2. Renal lymph disorder took an obviorsly effect on renal function and structure, which induced severe chronic renal lesion and chronic renal failure. The lesion was aggravated by the time of the disorder..
3. Renal lymph disorder induces tubulointerstitial fibrosis, and enhanced activation of the TGF-β_1/Smad singanling played a key role in the progress of the fibrosis and epithelial -mesenchymal transition .
4. Renal lymph disorder enhanced renal cells apoptosis. Bax/Bcl-2 pathway mediates apoptosis in this desease.
随着人民生活水平的提高及寿命的延长,慢性肾脏病(CKD)的发病率明显增高。慢性肾损害所致的尿毒症给病人带来了极大的痛苦及经济负担。因此探讨慢性肾损害的原发致病因素并采取早期干预措施有重大理论意义和临床实用价值。近年来,有关器官内淋巴管的超微结构及其循环障碍对组织影响的研究已有一些报道,尤其在脑、肝脏、胃肠等器官已取得了较大进展。然而,关于肾脏毛细淋巴管的超微结构以及循环障碍对肾脏影响的研究少见报道。早期曾有人研究过短时结扎犬肾门淋巴管后,对肾功能的影响,发现肾间质静水压增高,钠水排泄改变,尿量增加,随着时间延长,可出现肾间质的弥漫性损害。那么,肾脏淋巴循环和肾脏组织结构改变之间有着怎样的关系,又是通过何种机制起作用呢?至今尚无人深入研究。
我们早期的预实验发现结扎大鼠肾淋巴管后,尿蛋白显著增加,随着时间推移肾功能逐渐减退,组织病理出现以肾小管间质损伤为主的改变,包括上皮细胞崩溃脱落坏死,小管萎缩,正常结构破坏,肾间质纤维化,肾小球系膜细胞及基质增生等。我们推测肾淋巴循环障碍将激发肾脏纤维化与肾脏细胞凋亡的发生。我们知道肾间质纤维化是所有慢性肾脏疾病的共同特征,几乎是所有终末期肾病(ESRD)的最终转归,而肾小管上皮细胞-间充质细胞转化(EMT)是肾间质纤维化的重要发病机制之一。上皮细胞-间充质细胞转化过程的经典调控因子为转化生长因子-β超家族成员。SMAD家族蛋白的主要作用是介导TGF-β家族的信号。细胞凋亡又称程序性的细胞死亡,随着对细胞凋亡研究不断深入,发现Bcl-2家族是在细胞凋亡中有重要作用的一类蛋白质。Bcl-2家族的表达和调控是影响细胞凋亡的关键因素之一,在细胞凋亡信号转导途径中发挥重要作用。Bcl-2和bax分别是bcl-2家族中最有代表性的抑制凋亡和促进凋亡基因,并且bax是bcl-2活性的主要调控因子。尽管,对肾间质纤维化和细胞凋亡的研究已较深入广泛,但肾淋巴循环障碍是否引起肾间质纤维化和细胞凋亡,TGF-β_1/smad途径和bax/bcl-2途径在其中的作用如何却仍不清楚。
本课题吸收国内外研究的最新成果,结合国内外肾脏病防治的迫切需要,通过动物试验,用结扎肾淋巴管的方法建立肾淋巴循环障碍大鼠模型,通过观察肾组织病理及功能变化用以证实肾淋巴循环障碍所导致的肾脏损害,通过对TGF-β/SMAD途径和Bcl-2/bax途径的相关分子蛋白的检测,阐明其肾间质纤维化及肾细胞凋亡的发生机制,为临床越来越多的不明原因的慢性肾损伤寻找新的原发致病因素。
研究方法:
1.鼠肾起始淋巴管的分布及形态学观察
1.1 Wistar大鼠20只,雌雄不限。常规麻醉、手术开腹。每只动物取肾组织用免疫荧光双标法,即淋巴管标记物Podoplanin和血管内皮标记物CD34分别标记淋巴管和血管,应用Leica荧光显微镜在不同的通道(绿光激发光波长543nm;红色激发光波长633nm)分别观察并拍照,以观察鼠肾起始淋巴管在肾脏分布。
1.2肾脏超微结构观察:在皮质,髓质及皮髓交界区取组织块置3%戊二醛固定液内,用透射电镜观察淋巴管超微结构,包括毛细淋巴管的一般特征、内皮细胞的连接、质膜小泡的形态和分布。
2.对大鼠肾脏结构功能的观察
2.1将大鼠分为两组:模型组和对照组,每组30只。用结扎肾淋巴管的方法建立了肾淋巴循环障碍大鼠模型。各组大鼠分别于术后第7、14、28、56天各处死6只。在处死前1日留取24小时尿标本,处死日留取血标本。尿蛋白和血清肌酐由全自动分析仪检测。Ccr按公式:尿肌酐浓度×每分钟尿量(ml)/血肌酐浓度计算,并以体重校正。以观察不同时间段大鼠尿蛋白、肾功能改变。
2.2肾脏组织经固定、脱水、石蜡包埋,切片3μm厚度,做PAS、Masson染色。在光镜下观察肾脏病理改变,并根据小管损伤,小管间质纤维化和肾小球硬化标准作小管损伤指数,小管间质纤维化指数(TIFI)和肾小球硬化指数(GSI)的半定量分析。
2.3留取约1mm~3大小肾皮质经固定、包埋,切片厚50nm,观察肾脏超微结构改变。
3.淋巴循环障碍促使肾间质纤维化的研究
假手术组和模型组大鼠分别于术后第7、14、28、56天处死。留取肾组织标本提取组织蛋白、mRNA基因和制作石蜡切片。运用Real-time PCR、Western Blot和免疫组化检测检测TGF-β_1、Smad2/3、P-smad2/3在肾脏组织的表达。
4.肾淋巴循环障碍促使肾细胞凋亡的研究
各组大鼠分别于术后第1、7、14、28天各处死6只,留取肾组织标本提取组织蛋白、mRNA基因和制作石蜡切片。运用Real-time PCR、Western Blot和免疫组化检测检测Bax、Bcl-2、Caspase3在肾脏组织的表达,用肾脏细胞原位凋亡(TUNEL)的方法检测肾脏凋亡细胞。
结果:
1.鼠肾起始淋巴管的分布及形态学观察
1.1经免疫荧光双重染色,光镜下可见毛细血管呈红色、毛细淋巴管呈绿色,区别明显。淋巴管形态不规则,管腔大,壁薄。淋巴管主要位于富含结缔组织区,大鼠肾脏皮质淋巴管明显多于髓质。
1.2电镜下见毛细淋巴管的形状极不规则,毛细淋巴管的管壁较薄,毛细淋巴管内皮细胞无窗孔,基膜很薄,不连续或缺如,可见细胞内通道样结构,淋巴管内皮细胞中富含细胞器和质膜小泡。内皮细胞以重叠连接为主,在内皮细胞的连接处可见有粘着装置。毛细淋巴管的内皮细胞中含有极其丰富、大小不等的质膜小泡。质膜小泡多数游离于细胞质内。
2.对大鼠肾脏结构功能的观察
2.1模型组大鼠于术后第1周即出现明显蛋白尿(P＜0.05),随时间的延长而加重;模型组大鼠血肌酐水平在第2周时开始升高,(P＜0.05),且随术后时间延长,有逐渐增高趋势;模型组Ccr水平从术后第2周起明显下降,以后随着术后时间的延长,不断下降。
2.2模型组大鼠出现明显的组织病理改变,肾小管扩张明显,伴上皮细胞空泡变性,部分小管出现上皮细胞崩溃脱落,小管结构不完整。随术后时间延长出现肾小管萎缩,间质面积增宽,小管和间质结构消失,其中可见大量纤维组织增生。肾小球系膜区可见系膜细胞及基质增生。小管损伤指数,小管间质纤维化指数(TIFI)和肾小球硬化指数(GSI)较对照组明显增高,并随病程逐渐进展。
2.3电镜下观察模型组于造模后1周即出现肾小管上皮细胞、肾小球足细胞水肿,胞质内空泡数目增加,线粒体肿胀,部分嵴消失部分线粒体嵴消失呈空泡状;足突融合、变平;系膜增生插入,基质呈高电子密度。
3.淋巴循环障碍促使肾间质纤维化的研究
3.1免疫组化显示,模型组大鼠在小管上皮及系膜区的TGF-β_1、Smad2/3表达明显增强,在小管上皮细胞更为明显。ColⅠ在肾间质及肾小管上皮细胞的表达明显增高,而肾小球无明显变化。p-Smad2/3主要表达于肾小管上皮细胞,在肾小球系膜细胞的细胞核内也可见较强的p-Smad2/3的表达。
3.2 Western印迹检测发现模型组TGF-β_1、Smad2/3的蛋白表达量较对照组明显增加(P＜0.05),在第1周开始就有p-Smad2/3蛋白表达,第2周后持续增高,以后维持在较高水平(P＜0.05)。
3.3 Real-time PCR的检测显示ColⅠ、TGF-β_1、Smad2、Smad 3的mRNA均于术后第1周开始增高,分别为同期对照组的4.5、1.6、1.7、1.8倍(P＜0.05)。
4.肾淋巴循环障碍促使肾细胞凋亡的研究
4.1应用TUNEL检测发现,在模型组大鼠阳性着色细胞显著增加,且细胞凋亡主要发生在扩张或萎缩的肾小管;模型组的凋亡指数在术后第一天开始升高(p＜0.05),在第14天达到高峰(p＜0.01)。
4.2免疫组化提示,模型组的Bax和caspase-3表达明显增强,主要位于小管间质,尤其在远端小管。相反,Bcl-2的表达明显减弱。
4.3 Western blot分析显示,模型组大鼠Bax蛋白表达水平明显增加,在术后第14天达到高峰(p＜0.01),以后维持缓慢增高趋势。然而Bcl-2蛋白表达从术后第一天开始明显下降。由此,Bax/Bcl-2比值明显增高。总caspase-3表达明显高于对照组(p＜0.01),且其活化片段(17-kDa)表达也明显增高。
4.4 Real-time PCR显示在所有时间段,模型组大鼠bax和caspase-3的mRNA表达显著增高,而bcl-2 mRNA表达却明显下降(p＜0.05)。同样Bax/Bcl-2比值也较对照组明显增高。
结论:
1.大鼠肾脏淋巴系统内大量质膜小泡和通道样结构的存在,正是肾脏转运大分子物质和组织液进入淋巴循环的生理基础。
2.肾淋巴循环障碍对肾脏结构和功能有着明显的影响,肾脏功能减退和结构损害随循环障碍时间的延长而加重。
3.在肾淋巴循环障碍导致的小管间质纤维化过程中,TGF—β_1/Smad途径被高度激活,促进着上皮细胞的转型,在纤维化过程中起着积极作用。
4.在肾淋巴循环诱导的肾脏功能和结构改变的过程中,细胞凋亡显著增加,其中Bax/Bcl-2途径发挥了积极作用。
Background and objective:
Recent surveys have revealed that the prevalence of chronic kidney disease (CKD) is surprisingly high in the general population. Most patients with CKD develop end-stage renal failure, which requires costly treatment and unaffordable renal replacement therapy. Therefore, to find out the cause of chronic renal damage and to develop the effective treatment seemed to be emergency. Renent year, the study of lymph ultrastructure in some organs and their circlulation disorders have been done, especially in brian, liver, and stomach intestine. However, there is very few about renal lymph ultrastructure and the effect of its disorder on renal function and structure. The knowledge on renal lymphatics is very limited and their importance is almost always neglected. The role of lymph in keeping the balance between reabsorption of fluid from the tubules and uptake of reabsorbed fluid by the peritubular capillaries is highlighted. Early papers demonstrated that acute ligation of hilar lymphatic ducts has a significant effect on renal function, such as changes of sodium excretion, urine volume. As long as the time goes by, it appears diffuse lesion in renal interstitium. Therefore, what is the relationship between renal lymph circlulation and renal structure, what is the exact mechanism to interfere with? There is nobody to study.
In our pre-study, we demonstrated that rats developed significant and progressive proteinuria and elevating serum creatinine, when renal hilar lymph ducts was ligated. These kidneys were characterized by tubular cell detachment, tubular cell necrosis, tubular atrophy or dilatation and deposition of ECM. Thus,we suggest that renal lymph disorder induces renal fibrosis and renal cell apoptosis. As we know, tubulointerstitial fibrosis is critical determinants of renal function, and is the common final way of end stage of renal disease. The renal epithelial -mesenchymal transition(EMT) is one of the important mechanism of tubulointerstitial fibrosis. It is well known that TGF-β1 plays a significant role in the progression of EMT. Smad proteins have been identified recently as important components of the TGF-β_1 signaling pathway. Apoptosis is so called programmed cell death, and rencent study revealed that Bcl-2 family members are significant to cell apoptosis. The expression and regulation of Bcl-2 family proteins play a key role in apoptotic signal transduction pathway. Proapoptotic Bax and antiapoptotic Bcl-2 are the typical genes in Bcl-2 family, and furthermore, bax is the chief active regulating factor of bcl-2. In this study, we established the rat model of renal lymph circlulation disorder to show the time course of renal lesions caused by lymphatic ligation ; and to reveal the exact mechanism of renal lesion through detect the molecule involved in TGF-β_1/ SMAD and Bcl-2 /bax pathway.
Methods:
1. The study of the distribution and morphologic feature of the intrarenal lymphatics
1.1 There are 20 female and male Wistar rats were involved in this study and nephridial tissue were obtained from each animal. Double immunofluorescence was applied to detect the lymphatic distribution by using Podoplanin and CD34. Using Leica fluorescence microscope , green light was detect under 543nm and red light under 633nm wavelength of light.
1.2 Observe of renal lymphatic ultrastructure :renal tissues were obtained from the cortex, medulla and junctional zone , and put in 3% glutaric dialdehyde stationary liquid for transmission electron microscope to observe the fine distribution and morphologic feature of the intrarenal lymphatics.
2. The study of rats renal function and structure
2.1 Animals were divided into two groups: control group and model group (30 rats in each group) .Establish the rat model of renal lymph circlulation disorder by renal lymph ligation. Six rats in each group were sacrifice at 7、14、28、56d after operation. Urine aliquot and blood preparation were obtained. Proteinuria and serum creatinine value were examed by automatic analyser .Creatinine clearance (Ccr) formula : urine creatinine×urine volume per minute/serum creatinine, corrected by body weight.
2.2 The renal tissue was examined by PAS and Masson staining after fixation, dehydration, paraffin imbedding. The assessment of tubulus injury index , TIFI and GSI was performed with the use of semiquantitative scores .
2. 3 Obtained 1mm~3 of renal cortex for fixation, embedding sectioning. The ultrastructure of renal tissue was detected by electron microscope.
3. The study of renal interstitial fibrosis induced by renal lymph circulation disorder
Rats in each group were sacrifice at 7、14、28、56d after operation. Nephridial tissue were obtained for extracting protein , mRNA and paraffin section. Real-time PCR, immunostaining and western blot techniques were applied to detect the expression of COLI , TGF-β_1, Smad2/3, P-smad2/3 in renal tissue.
4. The study of renal cell apoptosis induced by renal lymph circlulation disorder
Rats in each group were sacrifice at 7、14、28、56d after operation. Nephridial tissue were obtained for extracting protein , mRNA and paraffin section. Real-time PCR, immunostaining and western blot techniques were applied to detect the expression of Bax/Bcl-2, Caspase3 in renal tissue. Apoptotic nuclei were labeled using the terminal deoxynucleotidyltransferase dUTP nick-end-label (TUNEL) technique.
Results:
1. The study of the distribution and morphologic feature of the intrarenal lymphatics
1.1 Under immunofluorescence double staining, blood capillary showed schillerization, and lymphatic capillary showed green color. lymphatic capillary appeared anomalism, large lumina , thin wall and located in connective tissue region. Cortilymph obviously exceed medulla lymph in rats.
1.2 Under electron microscope, the basal lamina of lymphatic was discontinuity.A lot of lysosome and vesicles existed in the endothelial of capillary lymphatics. Many of the vesicles liberated in cytoplasm. The intercellular junctions of the renal lymphatics are held in close apposition.
2. The study of rats renal function and structure
2.1 Model group rats developed severe proteinuria and elevated serum creatinine and reduced creatinine clearance were observed.
2.2 Histomorphological changes appeared in ligated kidneys, characterized by tubular damage, tubulointerstitial fibrosis, and expansion of the mesangium. The index of tubular damage and tubulointerstitial fibrosis were remarkably aggravated in model group.
2.3 Under electron microscope, renal tubular epithelial cell andpodocyte showed edema; the number of vacuolus increased in intracytoplasm; mitochondrial crista disappeared; foot process got fusion; mesenterium got proliferation and insertion;ground substance became electron-dense
3. The study of renal interstitial fibrosis induced by renal lymph circlulation disorder
3.1 Immunohistochemical staining indicated that Overexpression of transforming growth factor-β_1 (TGF-β_1) , Smad2/3, COL I genes and proteins were detected in model group rats. These proteins were mainly expressed in renal tubulointerstitium .
3.2 Western blot showed protein expression of TGF-β_1 , Smad2/3 were increased in model group rats. The expression of p-Smad2/3 increased from week 1 and keep high level after then.
3.3 Real-time PCR revealed that the expression of Col I, TGF-β_1, Smad2, Smad 3 mRNA were up-regulation from week 1, as 4.5、1.6、1.7、1.8 folds as control group.
4. The study of renal cell apoptosis induced by renal lymph circlulation disorder
4.1 TUNEL exam showed that many positive staining cells in model group rats. cell apoptosis mainly occured in dilate and emarcide renal tubule. Apoptotic index elevated from day 1 in model group , peaked at day 14.
4.2 Immunohistochemical staining indicated that overexpression of Bax and caspase-3 in model group rats, mainly in renal tubulointerstitium. Oppositely, Bcl-2 was weakly.
4.3 Western blot showed protein expression of Bax, caspase-3 increased in model group rats, peaked at day 14. The active unit of caspase-3 (17 KD) was also elevated in model group. Icreasing bax to bcl-2 ratio were also detected in model group rats.
4. 4 Real-time PCR show that the expression of bax and caspase-3 were up-regulation in all time points. However, bcl-2 mRNA decreased in model group. Icreasing bax to bcl-2 ratio were also detected in model group rats. Conclusions:
1. The intercellular junctions of the renal lymphatics are held in close apposition by adhesion devices and a lot of lysosome and vesicles existed in the endothelial of capillary lymphatics, which are the base for renal to transport big molecular and fluids.
2. Renal lymph disorder took an obviorsly effect on renal function and structure, which induced severe chronic renal lesion and chronic renal failure. The lesion was aggravated by the time of the disorder..
3. Renal lymph disorder induces tubulointerstitial fibrosis, and enhanced activation of the TGF-β_1/Smad singanling played a key role in the progress of the fibrosis and epithelial -mesenchymal transition .
4. Renal lymph disorder enhanced renal cells apoptosis. Bax/Bcl-2 pathway mediates apoptosis in this desease.
引文
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