Subcellular and regional location of “brain” proteins BASP1 and MARCKS in kidney and testis

Proteins BASP1 and MARCKS are abundant in axonal endings of neurons. Similarly to brain-specific protein GAP-43, BASP1 and MARCKS are reversibly bound to the plasma membrane. These proteins control both actin polymerization and actin cytoskeleton binding to the membrane. Performing these functions, BASP1 and MARCKS take part in growth cone guidance during development and in neurotransmitter secretion in adults. These activities predetermine the pivotal role of BASP1 and MARCKS in learning and memory. BASP1 and MARCKS were also found in non-nerve tissues, in particular, in the kidney and testis. Evidently, the physiological roles of these proteins differ in different tissues. Correspondingly, their intracellular location and activities may not be similar to those in neurons. In this paper, we analyze subcellular fractions (cytoplasm and nuclei) of rat kidney and testis with the purpose of determining the intracellular location of BASP1 and MARCKS. Western blots demonstrated that in these tissues, as in the brain, both proteins are present in the cytoplasm of the cell. According to our immunohistochemical study, BASP1 and MARCKS are specifically distributed in the tissues studied. In kidney, both proteins are present in cells located in glomeruli. In the testicular tubules, BASP1 is mainly expressed at the late stage of spermatogenesis (in spermatids) and is preserved in mature spermatozoa, while MARCKS appears equally during all stages of spermatogenesis. MARCKS is not found in mature spermatozoa. The results indicate that study of functions of BASP1 and MARCKS in the kidney and in the reproduction system holds much promise.     

The relationship between autophagy and renal tissue injury in adriamycin nephropathy rats

Objective To observe the formation of autophagosome, the expression and distribution of autophagy-related protein LC3-Ⅰ, LC3-Ⅱ and Beclin-1 in adriamycin nephropathy rats at different pathological periods, to explore the relationship between autophagy and renal tissue injury, the occurrence of proteinuria, the progression of renal disease. Methods Sixty normal male SD rats were randomly divided into control group (n=30) and model group (n=30), the rats in model group were injected with adriamycin(6.5 mg/kg) via tail - vein for one time, while the rats in control group were injected with saline. Urine protein quantitation of 24 hour, the levels of serum albumin and total cholesterol were measured serially at the 2, 4, 6, 8, 10 weeks. The changes of kidney tissue pathology were detected after HE, PAS and Masson staining by light microscope. The formation of autophagy were detected by transmission electron microscopy, the localization and distribution of LC3-Ⅰ, LC3-Ⅱ and Beclin - 1 were detected by indirect immunofluorescence staining in kidney tissue, the autophagy - related proteins LC3-Ⅰ, LC3-Ⅱ and Beclin-1 expression was detected by Western blotting. Results In model group, urinary protein began to increase at the first two weeks, serum albumin decreased at the same time, and total cholesterol increased in the four weeks. There was a statistically significant difference compared with the control group (P＜0.01). The Scr and BUN were increased slightly at the four weeks in model group, and showed the deterioration of renal function after the eight weeks. There was a statistically significant difference compared with the control group (P＜0.01). Mesangial cell proliferation, mitochondrial swelling and foot process broadening and integration appeared early in the model group, while foot process disappearing and nuclear pyknosis were observed in the late by transmission electron microscope; Renal pathology gradually changed from mesangial proliferation to focal segmental glomerulosclerosis (FSGS) by light microscope. A low expression of autophagy was detected in renal tissue of control group rats by transmission electron microscopy and immunofluorescence microscope; in model group, with the progression of disease, the autophagy was significantly enhanced and maintained at a high level. With the progression of disease, the autophagy- related proteins LC3-Ⅰ, LC3-Ⅱ and Beclin-1 was significantly enhanced in the model group than the control group (P＜0.05). Conclusion Autophagy is involved in renal tissue injury and the occurrence of proteinuria, closely related to the progression of renal disease.     

免疫调节在特发性膜性肾病及足细胞损伤中的研究进展

特发性膜性肾病是一种常见的免疫介导的肾小球疾病，是成年人肾病综合征常见的病理类型之一，也是导致慢性肾脏病和终末期肾脏病的主要病因之一。足细胞是特发性膜性肾病蛋白尿发生的中心靶点，足细胞损伤是特发性膜性肾病进行性发展的重要因素，其中免疫复合物沉积及内环境发挥关键作用。本文对免疫调节在特发性膜性肾病及足细胞损伤中的发病机制进行综述。     

Effect of activating transcription factor 6 on the apoptosis of podocytes stimulated with palmitic acid

Objective To observe the effect of ATF6 on the apoptosis and proliferation of podocytes induced by palmitic acid (PA). Methods Podocytes were stimulated with different doses of PA for 24 h. The expression of cleaved-caspase3 was detected by Western blotting. The podocyte apoptosis was analyzed by flow cytometry (FCM), and the expression of ATF6 was tested by Western blotting and immunofluorescence staining. After the transfection of adenovirus siRNA against ATF6, the proliferation, the cell cycle and apoptosis of potocytes stimulated with PA were tested by MTT or FCM. Results The levels of cleaved-caspase3 and ATF6 of podocytes stimulated with PA were significantly increased by a dose-dependent manner compared with the control group (P＜0.05). The apoptosis of podocytes stimulated with PA was increased (P＜0.05). Compared with the podocytes stimulated with PA, the apoptosis of podocytes transfected by adenovirus siRNA against ATF6 with PA stimulation was significantly reduced (P＜0.05). The proliferation of podocytes transfected by adenovirus siRNA against ATF6 and stimulated with PA, however, was obviously increased compared with the podocytes stimulated with PA (P＜0.05). Conclusion ATF6 mediated the apoptosis of podocytes induced by palmitate acid.     

足细胞分子nephrin在高血压大鼠肾脏的表达及作用

目的探讨足细胞分子nephrin在自发性高血压大鼠（SHR）肾脏的表达及作用。方法监测不同时期SHR与京都大鼠（wistar-kyotorats,WKY）尾动脉收缩压（SBP）、尿β2-微球蛋白（β2-MG）、尿素氮（BUN）、血肌酐（SCr）水平;免疫组化、RT-PCR方法检测nephrin蛋白及mRNA的表达,观察肾脏的病理改变。结果与WKY组相比,SHR组SBP、β2-MG、BUN、SCr升高,nephrin蛋白及mRNA表达量下降,且nephrin含量与尿β2-MG呈负相关。SHR组肾脏发生病理改变。结论足细胞分子nephrin在SHR肾小球表达减少,可能是导致足细胞裂隙膜损伤,引起尿β2-MG排泄水平增加,肾功能受损,肾脏病理改变的基础。     

Genetics of idiopathic nephrotic syndrome

Nephrotic syndrome (NS) is a pathological entity characterized by massive proteinuria and has diverse etiology. Although it is one of the most common renal diseases in children, the etiological factors responsible for idiopathic NS/FSGS remain largely unknown. Previous studies had implicated a variety of factors including genetic factors, although NS is generally regarded as a sporadic disease. Familial cases of NS have however been reported periodically, and both autosomal dominant and recessive forms have been identified. Studies of familial NS /FSGS have led to the discovery of several genes that are expressed in podocytes and are associated with proteinuria. These discoveries have shifted the focus from glomerular basement membrane (GBM) to recognition of the central role of podocytes in maintaining glomerular perm selectivity and pathogenesis of NS/FSGS. Associations with various genes (NPHS1, ACTN4, NPHS2, WT-1) and linkage to several chromosomal regions (such as 19q13,11q21,11q24) have been reported in patients with familial NS/FSGS.     

尿足细胞标志蛋白Podocalyxin检测在糖尿病肾脏损害程度评估中的意义

目的探讨尿液足细胞表面标志蛋白Podocalyxin(PCX)检测在糖尿病肾脏损害程度评估中的意义。方法选取97例2型糖尿病患者及30例健康体检者(正常对照组)。按照尿清蛋白排泄率(UAER)及血清肌酐(SCr)水平,将97例糖尿病患者分为4组:Ⅰ组,UAER<30 mg/24 h,SCr≤135μmol/L,27例;Ⅱ组,UAER为30～299 mg/24 h,SCr≤135μmol/L,29例;Ⅲ组,UAER≥300 mg/24 h,SCr≤135μmol/L,22例;Ⅳ组,UAER≥300mg/24 h,SCr>135μmol/L,19例。采用酶联免疫吸附试验(ELISA)法检测5组受检者尿液中PCX水平,分析尿液中PCX与UAER、尿蛋白定量的关系。结果与正常对照组比较,Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组糖尿病患者糖化血红蛋白(HbA1c)、尿中PCX、UAER显著升高(P<0.05),Ⅱ组、Ⅲ组、Ⅳ组SCr、尿蛋白定量亦显著升高(P<0.05);且随着糖尿病肾病的进展,Ⅰ组、Ⅱ组、Ⅲ组和Ⅳ组尿液中PCX分别为(5.8±1.0)μg/L、(15.3±4.3)μg/L、(28.9±9.0)μg/L和(23.3±10.6)μg/L,Ⅰ组、Ⅱ组、Ⅲ组组间两两比较,差异均有统计学意义(P<0.05),Ⅲ组与Ⅳ组比较差异无统计学意义(P>0.05)。相关分析显示,尿液中PCX与UAER、尿蛋白定量均呈正相关(r值分别为0.78和0.58,P均<0.01)。结论尿液PCX检测可反映糖尿病肾病的进展。     

表没食子儿茶素没食子酸酯对高糖环境下体外小鼠足细胞活性氧的影响

目的　观察不同浓度表没食子儿茶素没食子酸酯（EGCG）对高糖造成氧化应激状态下体外小鼠足细胞损害的作用并探讨其机制。 方法　以高糖（25 mmol/L）培养的小鼠足细胞为研究对象,维生素E培养为对照。首先以MTT法检测细胞活力,再在激光共聚焦显微镜下以CM-H2DCFDA荧光探针观察不同浓度EGCG（0.2、10、100 μmol/L）刺激足细胞6、12、24 h后活性氧（ROS）生成,并以流式细胞仪定量分析ROS平均荧光强度。RT-PCR法检测足细胞内ROS产生的主要通路NADPH氧化酶各亚基mRNA（ph22phox、p47phox、p67phox）的表达。 结果　高糖刺激下6 h,足细胞ROS生成显著增加（P < 0.01）。正常糖组和甘露醇组培养12 h ROS生成无显著增加（P > 0.05）。EGCG 0.2 μmol/L作用6 h可显著降低高糖环境下体外小鼠足细胞ROS水平（P < 0.01）。与高糖组比较,EGCG（100 μmol/L）显著减少NADPH氧化酶亚基p22phox和p67phox mRNA表达（均P < 0.05）。与维生素Ｅ组比较,EGCG（0.2 μmol/L）和维生素E（0.2 mmol/L）协同作用组显著减少p47phox mRNA表达（P < 0.05）。 结论　EGCG能缓解高糖环境下体外足细胞氧化应激状态,对高糖培养下足细胞有保护作用。     

高糖诱导足细胞发生上皮-间叶细胞转分化

目的 通过观察高糖是否引起小鼠足细胞发生上皮-间叶细胞转分化现象,探讨糖尿病肾小球损伤的可能机制。 方法 以小鼠永生化足细胞株为研究对象,予不同浓度葡萄糖（12.5、25、50 mmol/L）处理该细胞36 h,并设低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理组为对照组。采用蛋白免疫印迹和间接免疫荧光染色方法检测α平滑肌肌动蛋白 （α-SMA）、迁连蛋白（FN）、CD2相关蛋白（CD2AP）和Wilms’肿瘤1基因（WT-1）蛋白的表达。 结果 低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理条件下小鼠足细胞表达WT-1、CD2AP,基本不表达α-SMA和FN;而予不同浓度葡萄糖处理36 h后,足细胞中α-SMA和FN表达水平呈剂量依赖性上调（P < 0.05）。间接免疫荧光染色结果也显示,与低糖组相比,高糖组中 α-SMA阳性的足细胞比例显著增加（P < 0.05）。同时,蛋白免疫印迹结果还表明高糖可呈剂量依赖性方式下调WT-1和CD2AP的表达（P < 0.05）。 结论 在高糖条件下,足细胞发生向间叶细胞表型的转分化可能是引起足细胞功能失调,进而引起糖尿病肾小球损伤发生的作用机制之一。