Fig. 5.

Chronic albumin overload slows degradation kinetics. Opossum kidney (OK) cells were pretreated with 20 mg/mL apical fatty acid-free BSA (FAF-BSA) for 72 h or 20 mg/mL Fraction V BSA (FrxV-BSA) or palmitate-loaded BSA (Palm-BSA) was added basolaterally for 3 h. After being washed, cells were incubated with ¹²⁵I-labeled albumin (¹²⁵I-albumin) for 15 min. A: total ¹²⁵I-albumin internalized under each condition (normalized to control; mean ± SD of 5 experiments). *P ≤ 0.05 and ****P ≤ 0.0001 by Kruskal-Wallis test (Dunnett’s multiple-comparisons test). B: cells incubated with FAF-BSA, FrxV-BSA, or Palm-BSA as in A were solubilized, and equal protein loads were Western blotted with anti-megalin antibody. A representative blot is shown, and normalized intensities (means ± range) from 2 independent experiments are plotted. C: cells treated as above were returned to culture after internalization of ¹²⁵I-albumin. Apical and basolateral medium from individual filters was collected, and cells were solubilized after 10, 20, or 30 min. Trichloroacetic acid-soluble ¹²⁵I-dpm are plotted as the percent total for each condition. The mean ± SD of 3 experiments is plotted. *P ≤ 0.05 and **P ≤ 0.01 by two-way ANOVA (Dunnett’s multiple-comparisons test).