Kinetics of endocytosis in renal proximal tubule studied with ruthenium red as membrane marker

Abstract

This study was performed to determine the membrane area of the different compartments involved in endocytosis in rat kidney proximal tubule. This enables a direct estimation of the kinetics of the membrane turnover in the subprocesses of endocytosis and recycling of membrane constituents. To accomplish this, cross-sectioned invaginations have to be distinguished from endocytic vesicles. To obtain a continuous heavy staining of the apical plasma membrane, we have developed a sandwich-staining technique involving initial staining with ruthenium red, osmium, and thiocarbohydrazide functioning as a molecular bridge between osmium molecules. The distribution of invaginations, noncoated and coated endocytic vesicles, as well as the surface density of invaginations, small endocytic vesicles, large endocytic vacuoles, lysosomes, and dense apical tubules are determined in segment 1 (S1) and 2 (S2) of the proximal tubule. This has shown that invaginations constitute most [54% (S1 and 62% (S2)] of the membrane in small membrane-bounded structures in the apical cytoplasm. No morphological characteristics enabled direct differentiation between cross-sectioned invaginations and small endocytic vesicles, but a method for correct identification of 72% of all invaginations and 82% of all small endocytic vesicles is presented. Using the surface densities of various compartments together with previous data on the internalization of membrane markers, we developed a kinetic model enabling calculation of the velocity of membrane internalization and subsequent recycling. The velocity of internalization is 6.4 X 10(-3) microns2.micron-3.s-1, corresponding to internalization of a membrane area equivalent to the entire surface of invaginations within 78 s. The velocity of membrane transfer from the vacuolar compartment to dense apical tubules and of recycling from dense apical tubules to the luminal membrane is 6.2 x 10(-3) microns 2.microns-3.s-1. The entire membrane area equivalent to the surface of the vacuolar compartment and dense apical tubules is transferred in 43 and 92 s, respectively. The amount of membrane that surrounds lysosomes is transported from the vacuolar compartment within 23 min.