J Am Soc Nephrol 9: 1767-1776. 1998 Characterization of a Kidney Proximal Tubule Cell Line, LLC-PK1 , Expressing Endocytotic Active Megalin RIKKE NIELSEN,* HENRIK BIRN,* S#{216}REN K. MOESTRUP,t MORTEN NIELSEN,t PIERRE VERROUST, and ERIK ILS0 CHRISTENSEN* *Department of Cell Biology, Institute of Anatomy, and tDepartment of Medical Biochemistry, University of Aarhus, Denmark; and Instit44t National de la Sante et de la Recherche M#{233}dicale U64, H#{244}pital Tenon. Paris, France. Abstract. Reabsorption and cellular handling of glomerular filtered vitamins, peptides, and hormones in the proximal tu- bule are essential, but thus far, poorly elucidated processes. The multiligand receptor megalin, initially described as a Hey- mann nephritis antigen and later identified as a member of the LDL receptor gene family, mediates reabsorption of several molecules, such as transcobalamin-vitamin B 2 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 endocy- totic apparatus. Furthermore, by immunoblotling and immuno- histo- and cytochemistry, megalin was identified in the endo- cytotic 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, th. endocy- tosed, iodinated RAP was located in endosomes and lysosomes in the apical part of the cells. Moreover, the LLC-PK 1 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 iran- scellular transport studies. The renal proximal tubule is responsible for reabsorption of hormones, vitamins, and peptides filtered in the glomerulus ( 1 ). Reabsorption and metabolism of these molecules are ex- tremely important for the avoidance of proteinuria and loss of essential vitamins. Vitamins such as 2 and folate are reab- sorbed in proximal tubule cells, and their homeostasis is reg- ulated by the cells (2-4). The exact cellular mechanisms un- derlying the regulatory processes of proximal tubule cells have not been fully elucidated. However, there is evidence that the receptor megalin mediates endocytosis of several molecules in the renal proximal tubule, e.g. , albumin and transcobalamin- vitamin B1, (2,5,6). Megalin was first described as a Heymann nephritis antigen (gp330) by Kerjaschki and Farquhar (7). The receptor is a 600-kD transmembrane protein belonging to the LDL receptor gene family (8). In addition to the ligands mentioned above, several others have been identified, including plasminogen activator inhibitor type-i (PAI-l) complexes, polybasic drugs, and receptor-associated protein (RAP) (9-15). RAP is a 40-kD protein situated in the endoplasmic reticulum and is thought to Received January 1 5, 1998. Accepted April 3, 1998. Correspondence to Dr. Erik Ils#{248} Christensen, Departnient of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C. Denmark. 1046-6673/09010- 1767$03.00/0 Journal of the American Society of Nephrology Copyright © 1998 by the American Society of NephroLogy act as a chaperone for the LDL receptor family proteins (16- 19). The distribution of megalin varies among the three seg- ments in the proximal tubule, but generally is located in mi- crovilli, coated pits, endocytotic vacuoles, lysosomes, and dense apical tubules (20). In addition to the renal tubule, megalin is situated in the retinal and inner ear epithelium, placenta, and yolk sac (6,21,22). Megalin has also been iden- tified in several cell lines, including L2 (origin: yolk sac) (23); MSV (origin: yolk sac) (24); F9 (origin: embryo) (25); and in a simian virus 40 (SV4O)-immortalized rat proximal tubule cell line (26). A stable proximal tubule cell line expressing megalin would be of considerable use for the examination of the events of binding, endocytosis, storage, and liberation to the circulation of vitamins and proteins in the proximal tubule, i.e. , transcel- lular transport mechanisms. The LLC-PK1 cell line is a porcine kidney cell line (27) possessing characteristics of the proximal tubule (such as Natdependent transport systems, enzymes located in the apical membrane including alkaline phosphatase, and -y-glutamyl transpeptidase) and an electric resistance of the monolayer of 1 27 f . cm2 that is similar to that of a leaky epithelia (28). Thus, the LLC-PK1 cell line could be a relevant tool for the examination of the functions of proximal tubule cells. This study characterizes the proximal tubular cell line with emphasis on its suitability for studies of binding, endocytosis, and transcellular transport. Transmission electron microscopy