engleski

Characterization of polyclonal antibodies raised to rat renal cortical endocytic vesicles

Reabsorption of filtered proteins from the renal proximal tubular (PT) fluid is mediated by endocytosis, a process that occurs via specialized intracellular organelles - endocytic vesicles (EV). The EV are also central to the vesicle recycling mechanism in the PT cells which continuously removes and replenishes various transporters and other membrane components in the brush-border membrane (BBM). Preparations of EV from the PT cells are usually contaminated with the non-endosomal cell organells. The aim of this study was to produce polyclonal antibodies to highly enriched preparations of the rat renal cortical EV that could be used for immunopurification of EV and/or other organelles from the PT cells. The EV were isolated from the rat kidney cortex homogenate by differential and Percoll density gradient centrifugation, characterized functionally and enzymatically, and used for immunization of the rabbits. As found by indirect immunofluorescence cytochemistry in 4 m thick cryosections of the fixed rat kidney, the immune sera brightly stained the BBM, EV and some others intracellular organelles in the PT cells. Western blot analysis of BBM and EV proteins showed that the sera labeled 9 protein bands of Mr 16-276 kDa. From these sera we immunoadsorbed 9 different antibodies to the respective proteins. The antibodies were characterized by immunoblotting and immunocytochemistry. The data showed that a) none of the polyclonal antibodies specifically labeled the EV, and b) an antibody to the 104 kDa protein strongly stained the BBM in the rat and human PT cells. Conclusion: Although none of the antibodies from the immune sera to purified rat renal cortical EV is useful for immunopurification of EV, some of the antibodies in these sera can be used to study the respective antigens in the rat and human PT cell organelles.

rat; kidney cortex; endocytic vesicles; antibody immunopurification

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