Involvement of cathepsins B and H in lysosomal degradation of horseradish peroxidase endocytosed by the proximal tubule cells of the rat kidney: II. Immunocytochemical studies using protein A-gold technique applied to conventional and serial sections.

Immunoelectron microscopic localizations of cathepsins B and H and injected horseradish peroxidase (HRP) in the lysosomal system of rat kidney proximal tubules were investigated by a protein A-gold technique. Kidneys were fixed by perfusion at fixed intervals after intravenous injection of HRP. At 5 to 15 minutes after the injection, the endocytic apparatus--including the apical vesicles, tubules, and vacuoles (endosomes)--were stained for HRP, but they were negative for cathepsins. Within 15 to 30 minutes after the HRP injection, HRP-containing endosomes were fusing with preexisting lysosomes. In the S1 segment, they accumulated in the apical cytoplasm and formed giant phagosomes, which increased markedly in number and size after 1 hour. These phagosomes were composed of a peripheral clear matrix and electron-dense inclusions. The clear matrix was stained heavily for cathepsins and HRP, whereas the electron-dense inclusions were consistently negative for cathepsins and HRP. The same results also were obtained after the double-labeling and serial sectioning techniques. The dense inclusions were fragmented gradually as the phagosomes decreased in size. After 3 hours, the size and number of phagosomes returned to their normal state (before the HRP injection). These results indicate that the endocytic apparatus of the proximal tubule cells does not contain cathepsins. Phagosomes are formed by the fusion of endosomes containing the internalized protein with the preexisting lysosomes. The degradation of HRP in giant phagosomes occurred rapidly. The coexistence of cathepsins B and H with the endocytosed HRP suggests that these cystein proteinases are involved in the degradation of protein in heterophagosomes of the proximal tubule cells.