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Polyclonal antibodies to rat renal cortical endocytic vesicles label proximal tubule cell membranes in the rat and human kidney

Proteins that are filtered in the renal proximal tubule (PT) are reabsorbed by endocytosis, a process which occurs via specialized intracellular organelles - endocytic vesicles (EV, endosomes). Due to imperfect methods, EV are hard to isolate without a significant contamination with other (intra)cellular membranes. The aim of this study was to produce specific polyclonal antibodies to the antigens in endosomal preparations that could be used either in immunoisolation experiments to get cleaner preparations of EV and other organelles or/and for specific labeling of the cell plasma membrane (PM) domains and intracellular structures in animal and human kidney. Endosomes were isolated from the rat kidney cortex homogenate by the established method (Sabolić I. and Burckhardt G., Methods Enzymol. 1990, 191:505), and characterized functionally and enzymatically. The final vesicle preparations exhibited high activity of the bafilomycin-sensitive proton-pump (V-ATPase) and minimal contamination with the brush-border (BBM) and basolateral membranes (BLM). Immune sera to the EV preparations were raised in rabbits. As found by indirect immunofluorescence cytochemistry in 4 m thick cryosections of the rat kidney cortex, the immune sera brightly stained the BBM and various intracellular organelles in the PT cells. In immunoblotting experiments, the sera strongly labeled 4 high (Mr 55-276 kDa) and 5 low molecular mass (16-36 kDa) protein bands. However, none of the bands was specific for EV. An antibody to the 104 kDa protein band was purified by immunoadsorption. This antibody strongly stained the BBM and subapical vesicles in PT cells of the rat and human kidney cortex. In the human kidney cortex, it also stained the BLM of some collecting duct cells. Thus, common antigenic domains exist in the cell membrane proteins in rat and human kidney tubules. Conclusion: Polyclonal antibodies to the rat renal EV failed to specifically label endosomes and, therefore, could not be used for better purification of these organelles. The antibodies, however, might be used in cell biology studies of the common membrane antigens along the rat and human nephron.

endosomes; immunoadsorption; immunocytochemistry; kidney cortex; rat

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