Nielsen R, Birn H, Moestrup S K, Nielsen M, Verroust P, Christensen E I
Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark.
J Am Soc Nephrol. 1998 Oct;9(10):1767-76. doi: 10.1681/ASN.V9101767.
Reabsorption and cellular handling of glomerular filtered vitamins, peptides, and hormones in the proximal tubule are essential, but thus far, poorly elucidated processes. The multiligand receptor megalin, initially described as a Heymann nephritis antigen and later identified as a member of the LDL receptor gene family, mediates reabsorption of several molecules, such as transcobalamin-vitamin B12 and albumin, in the proximal tubule. Consequently, a differentiated cell line of proximal tubular origin expressing megalin is an important requisite for examination of the above-mentioned processes. This study shows, using electron microscopy, that the cell line LLC-PK1, originating from the proximal tubule, maintained differentiated morphology and had a well developed endocytotic apparatus. Furthermore, by immunoblotting and immunohisto- and cytochemistry, megalin was identified in the endocytotic compartments of these cells. Megalin was situated mainly in the endosomes and in the dense apical tubules, but it was also identified in coated pits and in the brush border. The ability of megalin to mediate internalization and degradation of labeled receptor-associated protein (RAP) in a RAP-inhibitable manner was demonstrated. By autoradiography, the endocytosed, iodinated RAP was located in endosomes and lysosomes in the apical part of the cells. Moreover, the LLC-PK1 cells assembled in a monolayer with a hindrance toward diffusion of labeled mannitol, inulin, and dextran at a satisfactory level for the study of proximal tubule handling of smaller proteins. This study reveals a proximal tubule cell line expressing megalin in a functional manner well suited for binding, uptake, and transcellular transport studies.
近端小管对肾小球滤过的维生素、肽和激素的重吸收及细胞处理至关重要，但迄今为止，这些过程仍未得到充分阐明。多配体受体巨膜蛋白最初被描述为海曼肾炎抗原，后来被鉴定为低密度脂蛋白受体基因家族的成员，它介导近端小管中几种分子的重吸收，如转钴胺素 - 维生素B12和白蛋白。因此，一种表达巨膜蛋白的近端小管来源的分化细胞系是研究上述过程的重要条件。本研究通过电子显微镜显示，源自近端小管的LLC - PK1细胞系保持了分化形态，并且具有发育良好的内吞装置。此外，通过免疫印迹、免疫组织化学和细胞化学方法，在这些细胞的内吞区室中鉴定出了巨膜蛋白。巨膜蛋白主要位于内体和致密的顶端小管中，但也在被膜小窝和刷状缘中被鉴定出来。还证明了巨膜蛋白能够以RAP可抑制的方式介导标记的受体相关蛋白（RAP）的内化和降解。通过放射自显影，内吞的碘化RAP位于细胞顶端部分的内体和溶酶体中。此外，LLC - PK1细胞单层组装，对标记的甘露醇、菊粉和右旋糖酐的扩散有一定阻碍，达到了研究近端小管对较小蛋白质处理的满意水平。这项研究揭示了一种以功能方式表达巨膜蛋白的近端小管细胞系，非常适合用于结合、摄取和跨细胞运输研究。
