Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption

Abstract

The accumulation of fluorescein (FITC)-labelled bovine albumin was measured against the extracellular-fluid-phase marker FITC-inulin within confluent monolayers of the opossum kidney cell line OK. Fluorescence and electron microscopic pictures show that FITC-albumin is taken up by endocytosis and appears in a vesicular intracellular distribution. The uptake of FITC-albumin was quantified by measuring the cell-adherent fluorescence fluorimetrically. FITC-albumin uptake shows a time- and concentration-dependent saturation kinetics in contrast to the non-saturable FITC-inulin uptake, and exceeds the latter more than tenfold at low concentrations. Half-maximum saturation occurs at 20-30 mg/l. Initial FITC-albumin uptake/mg protein is stimulated by cell maturation, being six-to sevenfold higher in the confluent than in the subconfluent state, while FITC-inulin uptake is unchanged. Both an elevation of ambient osmolality to 600-750 mOsm/kg and disruption of the cytoskeleton by cytochalasin B (0.1 mmol/l) reduce initial FITC-albumin uptake by 50%-60% in a non-additive fashion. Albumin endocytosis is reduced both in acidic (pH 5.4) and alkaline (pH 8.4) medium, but does not depend on extracellular sodium, calcium or chloride. High concentrations of fetal calf serum or unlabelled albumin reduce FITC-albumin endocytosis dose-dependently. The present study is the first to investigate both the protein uptake and the fluid-phase endocytosis in a cultured proximal tubular cell line, using these cells as a model system-for proximal tubular protein reabsorption.