Establishment of an in vitro co-culture system for the renal glomerular filtration barier.

肾脏生成尿液的第一步是通过肾小球滤过屏障进行肾小球滤过，防止蛋白质渗漏到尿液中。为了研究肾小球滤过屏障超微结构受损的肾小球疾病的病理生理，有必要建立可靠的模型系统来再现该屏障的结构和功能。滤过屏障由肾小球毛细血管内皮细胞、肾小球基底膜和足细胞（又称肾小球上皮细胞）三部分组成。本研究旨在利用细胞培养系统建立这样的模型。我们利用国外合作者提供的条件永生化小鼠足细胞细胞系，研究了调控足细胞形态发生（过程形成和细胞-细胞接触形成）的信号转导通路，特别参考了rho家族小GTP…More ases (RhoA, Rac1, Cdc42) （Kobayashi N, 2002; Kobayashi et al., 2004; Gao et al., 2004; Gao et al.，手稿正在准备中）。由于在体内肾小球中，足细胞会形成细胞类型特异性的细胞间连接，即狭缝膜，因此足细胞间细胞间连接的形成被认为是滤过屏障形成的关键步骤。我们已经证明，通过用福斯克林抑制RhoA，培养的足细胞之间可以诱导紧密的细胞间接触。生化方法揭示了rho家族的其他成员，Rac1和Cdc42在细胞-细胞接触形成过程中被激活。在体外建立肾小球滤过屏障，似乎不太可能绝对需要共培养系统（足细胞+内皮细胞）。我们还表明，RhoA及其下游效应物ROCK的药理抑制可以促进培养足细胞的过程形成。按照本研究开始时的计划，我们仍在尝试建立肾小球内皮细胞细胞系。少

The initial step of the urine generation in the kidney is the glomerular filtration through the glomerular filtration barrier, which prevents protein leakage into the urine. In order study the pathophysiology of glomerular diseases, where the ultrastructure of the glomerular filtration barrier is damaged, it is necessary to establish a reliable model system to reproduce the structure and function of the barrier. The filtration barrier is composed of three components, i.e. the glomerular capillary endothelial cell, the glomerular basement membrane, and the podocyte, also known as the glomerular epitheliaI cell. This study has aimed to establish such a model using cell culture system.With the conditionally immortalized mouse podocyte cell line which is kindly provided by our foreign collaborators, we have studied the signal transduction pathway regulating the podocyte morphogenesis (both process formation and cell-cell contact formation) with special reference on the RHO-family small GTP … More ases (RhoA, Rac1, Cdc42) (Kobayashi N, 2002 ; Kobayashi et al., 2004 ; Gao et al., 2004 ; Gao et al., manuscript in preparation). Since in in vivo glomeruli, podocytes develop a cell-type specific intercellular junction, slit membrane, the formation of intercellular junctions between podocytes is thought to be the critical step in the development of the filtration barrier. We have shown that tight cell-cell contacts are induced between cultured podocytes by inhibition of RhoA with forskolin. Biochemical approach revealed the activation of other members of the RHO-family, Rac1 and Cdc42, during this cell-cell contact formation. It seems unlikely that a co-culture system (podocytes + endothelial cells) is absolutely necessary to establish the glomerular filtration barrier in vitro. We have also shown that pharmacological inhibition of RhoA and its downstream effector, ROCK, enhances process formation of the cultured podocytes. As planned at the beginning of this study, we are still trying to establish cell lines of renal glomerular endothelial cells. Less