Figure 9.

Long-term stimulation of fluid flow in the collecting duct by high K⁺diet decreases Piezo1-mediated Ca²⁺influx in principal cells.A, the averaged time-courses of [Ca²⁺]_i changes upon application of a selective Piezo1 agonist, 20 μM Yoda-1 (shown with the black bar on top) in individual principal (PCs, black) and intercalated (ICs, gray) cells within split-opened area of the collecting ducts isolated from mice fed a high potassium (5% K⁺) diet for 1 week. Number of individual cells is shown. Six different collecting ducts from three different mice were used for analysis. B, the summary graph comparing the magnitudes of Yoda-1-mediated [Ca²⁺]_i elevations calculated as the difference in [Ca²⁺]_i values before (time point 1), at the beginning (time point 2), and at the end (time point 3) of Yoda-1 application in individual PCs and ICs from the conditions in A. Bars and whiskers represent SE and SD, respectively. Mean and median values are denoted with a dot and a line, respectively. Dashed lines represent respective average values in control (untreated) conditions. # - significant decreases (p < 0.05, one-way ANOVA with post hoc Tukey test) versus respective control values shown in Figure 2C. C, representative high magnification confocal images of split-opened collecting ducts probed with anti-aquaporine 2 (pseudocolor red) and anti-Piezo-1 (pseudocolor green) isolated from mice kept on regular (0.9% K⁺) and high potassium (0.9% K⁺) diets. Nuclear DAPI staining is shown with pseudocolor blue. Images were rotated to achieve vertical position of the collecting ducts. Side areas with no data are separated by thin white dashed lines. Respective Z-stacks (XZ planes) in the area depicted by the thick white dashed lane are shown below. “a” and “b” denote apical and basolateral sides, respectively. D, summary graph comparing nuclear eccentricity in principal (PCs) and intercalated cells (ICs) in freshly isolated split-opened collecting ducts from mice kept on regular (0.9% K⁺) and high potassium (5% K⁺) diets. Four different collecting ducts from two different mice were used for analysis. For each individual cell, nuclear eccentricity was assessed as the ratio of the maximal height to maximal widths of DAPI fluorescence. ∗- significant decrease (p < 0.05, one-way ANOVA with post hoc Tukey test) between experimental groups shown with lines on the top. DAPI, 4′,6-diamidino-2-phenylindole.