巢蛋白在足突广泛融合的肾小球中表达下调及与蛋白尿程度的相关性

目的 观察中间丝蛋白类的巢蛋白（nestin）在足突广泛融合的肾小球中的表达及其与足突病变动态过程和蛋白尿产生的关系。 方法 免疫组化法检测nestin在人正常肾组织及微小病变肾组织中的表达。构建氨基核苷嘌呤霉素肾病大鼠模型，应用免疫组化、荧光实时定量PCR、Western印迹法检测注射嘌呤霉素后第1、4、10、20天大鼠肾小球中nestin的分布与表达；电镜观察肾脏足细胞改变，测定尿蛋白量（24 h）。分析nestin的变化与蛋白尿的相关性。 结果 免疫组化显示nestin在人类微小病变肾小球中的表达较正常组织显著下调（0.93±0.08 比 1.65±0.12，P < 0.05）。在嘌呤霉素损伤足细胞早期，肾小球nestin的表达曾有一过性增加（mRNA和蛋白水平分别为对照组的1.23倍和1.48倍，P < 0.05），随后持续下降。nestin的mRNA水平在嘌呤霉素注射后第4天时降至对照组的35.8%；第10天时为对照组的12.1%（均P < 0.01）；病变好转后开始上升，恢复为对照组的65.8%（P < 0.05）。Western印迹检测nestin蛋白改变也有类似的趋势，嘌呤霉素注射后第4 天，nestin蛋白水平有所下降，为对照组的77.0%（P < 0.05）；至大量蛋白尿的第10天，nestin蛋白水平仅为对照组的58.0%（P < 0.05）；而随着病变的恢复，嘌呤霉素注射后第20天时nestin蛋白量恢复为对照的83.4%。Pearson相关分析结果显示，注射嘌呤霉素后nestin mRNA（r = －0.667，P < 0.05）及蛋白（r = －0.621，P < 0.05）表达与尿蛋白量(24 h)均呈负相关。 结论 在以足突广泛融合为特征的肾脏病变中，肾小球中间丝蛋白nestin表达显著减少，并与蛋白尿程度呈负相关，提示nestin可能参与了足细胞形态和功能的维持。     

肾缺血再灌注损伤大鼠模型足细胞损伤及机制研究

目的探讨肾缺血再灌注损伤(renal ischemia reperfusion injury,IRI)大鼠模型足细胞的损伤,并对其损伤机制进行初步研究。方法雄性SD大鼠42只,随机分为正常组(n=5)、假手术组(n=5)和模型组(n=32),模型组下设IRI后1h、6h、12h、24h4个时间点,每个时间点8只大鼠。通过阻断大鼠双侧肾脏血流构建肾IRI模型。采用全自动生化分析仪检测各组大鼠的肾功能,流式细胞仪检测血活性氧(reactive oxygen species,ROS)的变化,糖原染色(periodic Acid Schiff,PAS)了解肾脏病理损害,通过透射电镜观察足细胞损伤情况。结果在IRI大鼠模型中,我们发现了明显的足细胞损伤现象,即足细胞减少,足突消失,基底膜增厚。同时观察到,随着缺血再灌注后时间的延长,大鼠肌酐、尿素氮水平逐渐升高,红细胞中ROS荧光强度阳性率逐渐升高,肾小管出现不同程度的管腔扩张、变性与坏死,间质炎性细胞浸润、充血水肿等。相关性分析发现,红细胞ROS荧光强度阳性率与肾小管损伤评分、肌酐、尿素氮水平成正相关,与足细胞计数呈负相关。结论肾IRI早期足细胞就可出现明显的损伤,同时伴有血ROS荧光强度阳性率明显上升;血ROS的变化可能参与了肾IRI足细胞损伤及病理损害的过程。     

IQGAP1在调节足细胞细胞骨架重组中的作用

目的:研究支架蛋白IQGAP1在足细胞骨架重组中的作用。方法:用嘌呤霉素(PAN)刺激小鼠足细胞,实时定量PCR检测IQGAP1mRNA的表达,免疫印迹法检测足细胞IQGAP1蛋白的表达,激光共聚焦显微镜观察足细胞骨架F-actin分布,F-actin肌动蛋白评分系统(CFS)分析骨架重组;IQGAP1siRNA和IQGAP1质粒转染足细胞,分别观察下调和上调足细胞IQGAP1表达对嘌呤霉素诱导足细胞细胞骨架重组的改变。结果:嘌呤霉素刺激足细胞后IQGAP1mRNA和蛋白表达下调(P<0.05),足细胞F-actin分布发生明显重组;转染IQGAP1siRNA,嘌呤霉素刺激足细胞F-actin重组进一步加重;转染IQGAP1质粒,嘌呤霉素刺激足细胞F-actin重组减轻。结论:IQGAP1可能参与嘌呤霉素诱导的足细胞细胞骨架重组。     

二维平面足细胞相对密度测量法

目的：探索一种简便且临床实用的足细胞丢失评估方法。方法：应用免疫组化法对正常和阿霉素肾病大鼠的肾组织,以及正常肾组织和3种不同病理类型肾小球疾病患者肾组织进行Wilms′瘤蛋白免疫染色,采用手工计数和病理图像分析技术相结合的方法分别对单个肾小球内足细胞的绝对数以及单位面积肾小球内足细胞的相对密度进行测定,找出结果可靠的最低肾小球数目并检验不同测量人员所获结果的一致性。结果：在统计10个和15个大鼠肾小球时,阿霉素肾病组肾小球足细胞相对密度显著低于正常大鼠;无论统计的肾小球数目多少,两组大鼠肾小球足细胞绝对数差异无统计学意义。在统计5个和10个人类肾小球时,局灶节段性肾小球硬化组和膜性肾病组肾小球足细胞相对密度均显著低于正常组和微小病变组;局灶节段性肾小球硬化组肾小球足细胞绝对数显著低于正常组和微小病变组。人类肾小球足细胞相对密度与血肌酐（Scr）水平呈低度负相关,但与24h尿蛋白含量（24hUPQ）无相关关系;阿霉素肾病大鼠肾小球足细胞相对密度与Scr、24hUPQ无相关关系。两名检测者所获结果有高度一致性。结论：这种足细胞相对密度评估法简便、快捷和可靠,且所需肾小球数目少,有望在临床实践中推广。     

表没食子儿茶素没食子酸醋对高糖环境下小鼠足细胞增殖和凋亡作用

目的 观察作为绿茶主要活性成分的表没食子儿茶素没食子酸醋(EGCG)对高糖环境下足细胞增殖和凋亡的作用并探讨其机制.方法 将足细胞分为9组.组1:正常糖(5.6 mmol/L).组2:正常糖(5.6 mmol/L)+0.1 mmol/L H2O2.组3:DMEM高糖(25 mmol/L).组4:20 μmol/L EGCG+DMEM高糖(25 mmol/L).组5:2.0 μmol/LEGCG+DMEM高糖(25 mmol/L).组6:0.2 μmol/LEGCG+DMEM高糖(25 mmol/L).组7:0.2 mmol/L维生素E+DMEM高糖(25 mmol/L).组8:0.1 μmol/L EGCG+0.1 mmol/L维生紊E+DMEM高糖(25 mmol/L).组9:24.4 mmol/L甘露醇+5.6 mmol/L葡萄糖.采用溴脱氧核苷尿嘧啶(BrdU)酶联免疫吸附试验(ELISA)检测细胞增殖,在Hoechst染色共聚焦激光显微镜下观察不同浓度EGCG作用24 h对足细胞凋亡的影响,采用Annexin V-FITC法检测足细胞凋亡,CM-H2DCFDA荧光探针检测足细胞内活性氧(ROS)生成.结果 ①足细胞增殖:与组1比较,组2在刺激24 h时的足细胞增殖的光密度(D)450值无显著变化(P＞0.05),48和72 h时D450值均显著降低(P值均＜0.05).与组2比较,组7和组8高糖刺激24 h时的D450值显著升高(P值均＜0.05),组4、组5和组6无显著变化(P值均＞0.05);组4、组7和组8在刺激48、72 h时的Dm值显著升高(P值均＜0.01).②激光共聚焦显微镜下,凋亡细胞表现为较多的核固缩,DNA浓缩并向核膜靠拢,核质比减小,以及胞膜皱缩等早期凋亡形态学变化.组2、组3的足细胞凋亡较组1增加,组7明显少于组4、组5,组9较组2无明显增加.③不同浓度EGCG作用后,组4、组5、组8的早期凋亡细胞膜联蛋白V(+)PI(-)比例和坏死细胞膜联蛋白V(+)PI(+)V(+)比例均显著低于组2(P值均＜0.05);组4、组8的早期凋亡细胞膜联蛋白V(+)PI(-)比例均显著低于组7(P值均＜0.05).④与组2比较,组4 24 h时的ROS显著减少(P＜0.05),但组6无显著改变(P＞0.05);与组7比较,组4 24 h时的ROS显著减少(P＜0.05),6、12 h时无显著变化(P值均＞0.05).结论 EGCG(20 μmol/L)作用72 h促进高糖环境下足细胞增殖,EGCG降低高糖刺激下小鼠足细胞凋亡的作用可能是通过减少足细胞ROS的生成实现的.     

Invagination and infolding of podocytes in glomerular basement membrane in the cases of primary membranous nephropathy

Background  The ultrastructural findings of membranous nephropathy (MN) are well described. Recently, podocyte infolding in the glomerular basement membrane (GBM) has been observed to be a unique ultrastructural finding formed from diffuse spherical microparticles and microtubules in the GBM. However, these alterations of glomerular epithelial cells have not been well characterized in MN. Methods  We selected 126 renal biopsies of primary MN that were diagnosed by light microscopy and immunofluorescence. In these biopsies, we investigated the ultrastructural alterations of GBM and podocytes, especially the presence of podocyte invagination, podocyte infolding, and spherical microparticles in the GBM. Results  In 98 cases (77.8%) we ultrastructurally detected occasional invagination of podocytes in the GBM within or around electron-dense or lucent deposits in mainly stage II–III of MN. In 40 cases (31.7%), we found spherical microparticles in addition to the podocyte invaginations in the GBM. In our cases, spherical microparticles were divided into three types; podocyte infolding, cell debris and virus-like particle types. Only one case displayed numerous spherical microparticles (microspheres) that were probably caused by infolding of podocytes. These microspheres, about 80 nm in diameter, were covered by unit membrane, and were accompanied by similar-sized microtubules and protrusions of podocytes. The spherical microparticles in the other cases were associated with cell debris (n = 23) or virus-like particles (n = 16) and were not connected with podocytes. Conclusion  Podocyte invagination associated with subepithelial deposits was a common pathological finding of primary MN, especially stage II–III of MN. The spherical microparticles in GBM in the case of MN may be associated with not only podocyte infolding but also cell debris and virus-like particles. The spherical microparticles in GBM due to diffuse podocyte infolding was considered as a new pathology finding of the GBM and may appear to be a new glomerular disease entity termed podocytic infolding glomerulopathy.     

A case report of glomerulopathy-associated podocytic infolding in a patient with tumor lysis syndrome

A 59-year-old man underwent total gastrectomy and splenectomy for gastric cancer 14 months before admission. The pathological diagnosis was neuroendocrine carcinoma of the stomach. Eight months after the operation, systemic chemotherapy with irinotecan and cisplatin was started because of multiple metastases to lymph nodes. After two courses of chemotherapy, renal function continued to decline. Renal biopsy showed acute tubular necrosis with cast formation, where needle crystallization was found. These clinicopathological findings suggested that tumor lysis syndrome was the cause of acute renal insufficiency. Moreover, diffuse, global bubbling and focal segmental spike formation were revealed by periodic acid-silver methenamine stain in the glomerular basement membrane. Electron microscopy showed an infolding of the cytoplasm of podocytes into the basal basement membrane and spotty electron-lucent areas. These ultrastructural findings, but not epimembranous deposits, corresponded with the bubbling on PAM staining. The present case was a rare case of glomerulopathy associated with podocytic infolding, which was not associated with collagen disease but with tumor lysis syndrome.     

A nephropathy presenting the microparticles in the glomerular basement membrane in a patient of hepatitis B viral infection

We report here unique electron microscopy findings showing clusters of deposition of spherical and tubular microparticles in a glomerular basement membrane (GBM) of a 46-year-old Japanese male with membranous nephropathy. Another distinct feature was the deep infolding of podocyte membranes into the GBM. Light microscopy showed the ladder formation of the GBM suggesting membranous nephropathy, while the immunofluorescent examination was atypical for the absence of the global capillary deposition of IgG and C3. He had mild liver dysfunction with positive hepatitis B antigen. Antibodies to hepatitis B surface antigen reacted weakly on the GBM in the immunohistochemistry. We suspected the unique findings of this case might be related to the hepatitis B viral infection.