Ultrastructure of the Vacuolar Apparatus in the Renal Proximal Tubule Microinfused in vivo with the Cytological Stain Light Green

Abstract

To obtain insight into the basic mechanisms controlling endocytosis, we tested the effects of different perfusates containing the cytological stain light green on endocytosis and ultrastructure of the vacuolar apparatus in renal proximal tubule cells. Rat proximal tubules were microinfused in vivo for 2 min in the presence or absence of light green with the following solutions: (A) perfusates containing inorganic salts and (B) perfusates with organic components or protein. In other experiments, the tubules were first microinfused for 2 min with 0.9% NaCl in the presence or absence of light green, then 15 min later further microinfused with or without light green using either group A or B solutions in order to either aggravate or reverse possible changes. All infused tubules were fixed after 5 min with 1% glutaraldehyde and examined by electron microscopy. In tubules microinfused without light green, the endocytic vacuolar apparatus in the apical cytoplasm showed a normal ultrastructure. However, microinfusion of solutions containing light green in either inorganic salts or a low concentration of protein caused significant changes in the apical endocytic apparatus. Large endocytic vacuoles were absent, and invaginations and small endocytic vacuoles were decreased in frequency. On the other hand, the amount of dense apical tubules was significantly increased, and in some cells dense apical tubules had transformed into a cisternalike network. These changes were aggravated in tubules which received a second microinfusion of NaCl and reversed in tubules that received a second infusion of protein. Furthermore, in the tubules microinfused with light green using perfusates containing organic components or protein, the apical cytoplasm of proximal tubule cells showed an essentially normal endocytic apparatus. The present study demonstrates that microinfusion of renal proximal tubules with light green disrupted normal endocytic membrane trafficking and recycling. These changes could be prevented or reversed by microinfusion of solutions containing protein or organic components.