Figure 8.

Mice with transient receptor potential vanilloid type 4 (TRPV4) deletion in the renal tubule have increased H⁺-K⁺-ATPase-mediated intracellular pH (pH_i) recovery after intracellular acidification in freshly isolated split-opened collecting ducts. A: representative pseudocolor images (blue: acidic and red: alkali) of pH_i in a split-opened cortical collecting duct loaded with the pH-sensitive dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein at baseline (44), upon application of 40 mM NH₄Cl (25), immediately after NH₄Cl removal (40), upon recovery to baseline pH_i values (6), and following application of the ClC-K2 blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) to distinguish A-type and B-type intercalated cells (ICs) (1). A confocal micrograph of the same split-opened collecting duct probed with anti-aquaporin-2 (AQP2; pseudocolor red) to identify principal cells (PCs) is shown on the right. Examples of A-type (responding with alkalization to NPPB) and B-type (responding with acidification to NPPB) ICs and PCs (no change in pH_i to NPPB) are depicted with white, yellow, and orange arrows, respectively. Nuclear DAPI staining is shown as pseudocolor blue. B: summary graphs comparing the time course of pH_i changes in PCs (left), A-type ICs (middle), and B-type ICs (right) upon application of 40 mM NH₄Cl (shown by black horizontal line) and NPPB (shown by shaded horizontal bars) in cortical collecting ducts from TRPV4^fl/fl mice (red dots) and TRPV4^fl/fl-Pax8Cre mice (green dots) fed a low-K⁺ diet for 7 days. The time points shown in A are marked as 1–5. C: summary graphs of the recovery after acidification in individual PCs (right), A-type ICs (middle), and B-type ICs (right) within split-opened areas of collecting ducts from TRPV4^fl/fl mice (red dots) and TRPV4^fl/fl-Pax8Cre mice (green dots) fed a regular-K⁺ diet (0.9% K⁺) and low-K⁺ diet (<0.01% K⁺) for 7 days. The rate was calculated for each individual cell (shown as dots) as a linear slope of the initial pH_i recovery from the lowest pH_i value after 40 mM NH₄Cl removal. Numbers of cells in each group are shown. In average, six different collecting ducts from three different mice of each strain were used for each condition (1 female and 2 males or 2 females and 1 male). Cells from male and female are indicated as triangles and circles, respectively. *Significant changes (P < 0.05, one-way ANOVA with a post hoc Tukey’ test) vs. the respective values of TRPV4^fl/fl mice, as indicated by the horizontal bars.