Figure 3.

Altered PI(4,5)P₂ subcellular distribution and receptor-mediated endocytosis in mPTCs derived from Dent disease mouse models. (A) Representative confocal micrographs and quantification of PI(4,5)P₂⁺ structures (green) in Ocrl mPTCs (n ≈ 80 cells pooled from 3 mouse kidneys per condition; each dot representing the number of PI (4,5)P₂⁺ structures in a cell). (B) Representative confocal micrographs of Ocrl mPTCs immunostained with anti-PI(4,5)P₂ (green) and anti-EEA1 (red, early endosomes) and quantification (adjacent panel) of the number of PI(4,5)P₂/EEA1⁺ structures by confocal microscopy (percentage of total EEA1⁺ vesicles: n = 3 Ocrl^Y/+ and n = 4 Ocrl^Y/− randomly selected fields per condition, each containing approximately 15–20 cells). Insets: high magnification of PI(4,5)P₂/EEA1⁺ structures. (C,D) Ocrl and Clcn5 mPTCs were loaded with Alexa 488-BSA (green, 100 μg ml⁻¹ for 15 min at 37°C), fixed and analyzed by confocal microscopy. Quantification of the number of Alexa 488-BSA⁺ structures (n ≈ 150–250 cells pooled from 3 mouse kidneys per condition; each dot representing the number of BSA⁺ structures in a cell). (E) Ocrl mPTCs were loaded with Alexa 647-dextran 10 kDa (red, 250 μg ml⁻¹ for 30 min at 37°C), fixed and analyzed by confocal microscopy. Quantification of the number of Alexa 647-dextran⁺ structures (n ≈ 200–250 cells pooled from 3 mouse kidneys per condition; each dot representing the number of dextran⁺ structures in a cell). Nuclei counterstained with DAPI (blue). Scale bars: 15 μm in (A) and (B) and 10 μm in (C–E). Plotted data represent mean ± SEM. Two-tailed unpaired Student’s t-test, ^**P < 0.01, ^***P < 0.001 relative to Ocrl^Y/+or Clcn5^Y/+ mPTCs. ns: not significant.