Figure 5

To examine specific modes of endocytosis in CLVS1 podocytes, we evaluated the internalization of fluorescently labeled transferrin (clathrin-mediated endocytosis), 70,000 MW dextran (macropinocytosis), and albumin molecules (caveolae-mediated endocytosis). (A and B) Quantification of the number of internalized transferrin molecules per cell revealed a decrease in clathrin-mediated endocytosis in CLVS1-KO and homozygous H310Y-KI podocytes compared with controls that was unaffected by pretreatment with 1 μM dexamethasone. (n > 30 for each group, P < 0.0001, 1-way ANOVA.) (C and D) Macropinocytosis, as measured by the internalization of fluorescently labeled 70,000 MW dextran molecules, was unaffected by CLVS1 KO or H310Y KI (P = 0.9572 and P = 0.9604 respectively, 1-way ANOVA). However, treatment with dexamethasone did significantly increase macropinocytosis in all cell lines compared with vehicle-treated controls (P < 0.001 for all). (E and F) Caveolae-mediated endocytosis was comparable among CLVS1-KO, homozygous H310Y-KI podocytes, and controls when internalized albumin molecules were quantified, and this was unaffected by treatment with dexamethasone (n > 30 for each group, P = 0.993 and P = 0.1844 respectively, 1-way ANOVA). Error bars depict SEM in graphs. Scale bars = 12 μm. Each dot in the graphs represents the mean number of molecules per podocyte for a single well.