Characterization of a kidney proximal tubule cell line, LLC-PK1, expressing endocytotic active megalin.

Abstract

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.