Schiwek Daniel, Endlich Nicole, Holzman Lawrence, Holthöfer Harry, Kriz Wilhelm, Endlich Karlhans
Department of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany.
Kidney Int. 2004 Jul;66(1):91-101. doi: 10.1111/j.1523-1755.2004.00711.x.
Cell culture of podocytes has become an indispensable tool in the study of podocyte biology. To date, however, podocyte cell lines with stable expression of the crucial slit diaphragm protein nephrin and localization of nephrin to cell-cell contacts are not available.
Conditionally immortalized cells were grown from isolated glomeruli of mice, harboring the temperature-sensitive SV40 large T antigen. About 60 clonal cell lines were generated by limiting dilution.
Among 30 Wilm's tumor (WT)-1- and podocalyxin-positive cell clones, two cell clones stably expressed nephrin as assessed by reverse transcription-polymerase chain reaction (RT-PCR), Northern and Western blotting, and immunofluorescence. In addition, expression of the following podocyte proteins was demonstrated: NEHP1, FAT, P-cadherin, podocin, CD2AP, ZO-1 (alpha(-) isoform), Lmx1b, podoplanin, synaptopodin, cortactin, and vimentin. The nephrin-positive podocyte cell lines formed a monolayer with abundant cell-cell contacts. Transmission electron microscopy revealed formation of primitive foot process-like interdigitations and slit diaphragm-like junctions. Nephrin colocalized with F-actin at cell-cell contacts as demonstrated by immunofluorescence. Intriguingly, nephrin and actin-associated proteins (synaptopodin, CD2AP, and cortactin) were recruited to and accumulated at the entire cell margin only in confluent cells, but not in dispersed cells.
We present novel murine podocyte cell lines with stable expression of nephrin and abundant formation of cell-cell contacts, possessing several features of in situ podocyte cell-cell contacts. Furthermore, our data suggest that the accumulation of certain proteins in podocyte foot processes is linked to formation of cell-cell contacts.
足细胞的细胞培养已成为足细胞生物学研究中不可或缺的工具。然而，迄今为止，尚无稳定表达关键裂孔隔膜蛋白nephrin且nephrin定位于细胞间接触部位的足细胞系。
从携带温度敏感型SV40大T抗原的小鼠分离肾小球中培养条件永生化细胞。通过有限稀释法产生约60个克隆细胞系。
在30个肾母细胞瘤（WT）-1和足细胞标记蛋白阳性的细胞克隆中，通过逆转录聚合酶链反应（RT-PCR）、Northern和Western印迹以及免疫荧光评估，有两个细胞克隆稳定表达nephrin。此外，还证实了以下足细胞蛋白的表达：NEHP1、FAT、P-钙黏蛋白、足动蛋白、CD2AP、ZO-1（α异构体）、Lmx1b、血小板内皮细胞黏附分子、突触素、皮质肌动蛋白和波形蛋白。nephrin阳性的足细胞系形成了具有丰富细胞间接触的单层。透射电子显微镜显示形成了原始的足突样指状交叉和裂孔隔膜样连接。免疫荧光显示nephrin与F-肌动蛋白在细胞间接触部位共定位。有趣的是，nephrin和肌动蛋白相关蛋白（突触素、CD2AP和皮质肌动蛋白）仅在汇合细胞中被募集并聚集在整个细胞边缘，而在分散细胞中则没有。
我们展示了新的小鼠足细胞系，其稳定表达nephrin并大量形成细胞间接触，具有原位足细胞间接触的若干特征。此外，我们的数据表明某些蛋白在足细胞足突中的积累与细胞间接触的形成有关。
