Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry

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Abstract
The present study describes the intracellular distribution of Tamm-Horsfall protein (THP) in rat kidney. The localization was determined by immunoelectron microscopy using the protein A-gold technique. Various fixation and embedding protocols were evaluated for this purpose. Brief perfusion fixation (3 min) with 1% glutaraldehyde and embedding in a highly hydrophilic glycol methacrylate-polyester mixture were most appropriate for antigen-antibody recognition and structural preservation. The overall tissue distribution of THP was evaluated by indirect immunofluorescence microscopy; reaction was strong along the entire thick ascending limb of the loop of Henle (TAL) with enhanced fluorescence in the apical cytoplasm. On the electron microscopic level immunogold labelling was concentrated over numerous membrane-bound vesicles which form a compartment in the apical cytoplasm. The Golgi region was consistently labelled, whereas the plasma membranes revealed only sporadic labelling at the luminal side, and basolateral membranes were mostly unlabelled. Quantitative evaluation of the gold labelling, which was separately done for the inner stripe, outer stripe and cortical TAL, consistently showed the highest particle density in the apical cytoplasm. Middle and basal levels in the TAL cells were only moderately labelled. The results are discussed with respect to the current opinion which describes THP as a membrane glycoprotein. We speculate that the accumulation of THP in the apical vesicular compartment of TAL cells indicates a storage site of the protein, possibly prior to extrusion via exocytosis of the vesicle contents.