Fig. 10.

Gpr116 activation in A-ICs inhibits pH_i recovery. (A) Representative pseudocolor images (blue, acidic; red, alkali) of intracellular pH in a split opened collecting duct loaded with pH-sensitive dye BCECF at the baseline (1), upon application of 40 mM NH₄Cl (2), immediately after NH₄Cl removal (3), and recovery toward the baseline pH_i values (4). Confocal micrograph of the same split-opened CD probed with anti-AQP2 (pseudocolor red) is shown on the right. Examples of AQP2⁻ ICs are depicted with white arrows. Nuclear DAPI staining is shown in pseudocolor blue. Magnification: 40×. (B) Summary graph comparing the time course of pH_i changes in control (black) and p16 pretreated (100 µM for 40 min, red) ICs. Trace shows mean ± SEM for 65 control cells and 69 p16-treated cells. Each experimental condition is a summary from four collecting ducts from four different mice. Application of the NH₄Cl pulse is designated by the black bar above the trace. The time points shown in A are marked as 1 to 4. (C) Summary graph of H⁺ extrusion rate from ICs from the control and after pretreatment with p16 shown in B. The rate was calculated as a linear slope of pH_i recovery after 40 mM NH₄Cl application. Statistical analysis performed using Mann–Whitney test. *P < 0.05. Control vs. p16 *P < 0.0001.