Albumin and dextran uptake assay

For endocytosis experiments, mPTCs were cultured on collagen-coated polytetrafluoroethylene membranes (pore size of 0.4 μm). We previously showed that these cells are polarized with numerous microvilli, basolateral invaginations, and apical tight junctions (37). They express only PT-specific proteins and, in particular, the components of the receptor-mediated endocytic pathway, the PT brush-border enzymes (alkaline phosphatase and glutamyl-transferase), and the phloridzin-sensitive sodium-dependent glucose transport (SGLT2) on the apical membrane. However, these cells kept a flat morphology, and asymmetry of the apical compared with basolateral distribution of TRPV4 could not be demonstrated using immunostaining and confocal microscopy.

mPTCs were serum starved overnight and incubated with FITC-labeled albumin (0.5 mg/ml; Sigma-Aldrich) or FITC-labeled dextran (1 mg/ml; Sigma-Aldrich) in DMEM/F12 medium for 30 min at 37°C. The cells were washed with cold PBS five times and lysed in PBS with 0.1% Triton-X. The supernatant was used for fluorescence and protein assays. The intensity of FITC-fluorescence was measured by fluorophotometry. For the study of albumin endocytosis during cell stretching, cells were seeded on collagen I–coated flexible silicon chambers (ST-CH-10, B-Bridge) and placed in the stretching instrument (ST-140, B-Bridge). A uniaxial stretching of 20% was applied at a frequency of 0.3 Hz. At the end of the experiments, the cells were washed five times with ice-cold PBS, and fluorescence signals were immediately assessed using a fluorescence microscope.