FIGURE 4:

The DCT-specific puncta are KS-WNK1 dependent. (A) Immunofluorescence of mouse DCT from either WT or KS-WNK1^−/− (KO) mice maintained on LK diet for 10 d. [K+]_WB levels were similar for both WT and KS-WNK1 KO mice. DCTs were identified by NCC costaining. Pan-WNK1, WNK4, and SPAK antibodies detected puncta in WT mice, whereas puncta were nearly absent in KS-WNK1 KO mice. (n = 4 mice per genotype; scale bar = 10 μm in 1× images, 5 μm in 4× images). (B) Representative immunohistochemical staining of DCTs from KS-WNK1 KO mice maintained on either LK or HK diet for 10 d. Indicated with arrows are rare punctate structures that were detected in a small subset of DCTs with the pan-WNK1 antibody under both LK and HK conditions. (n = 3 mice per condition; DCT in 2.5× zoom indicated by a dashed line). (C–E) Comparison of WT and KS-WNK1 KO mice on LK and HK diets. KS-WNK1 KO mice exhibited dramatically reduced puncta abundance (C) compared with WT mice. These foci were positioned farther from the lumen (D) and demonstrated a normalization of diameter relative to WT (E; i.e., in KO mice, puncta diameter averaged 1.9 µM under both LK and HK conditions) (n = 3 mice per condition, and due to the scarcity of puncta more than 200 cells from 20 tubules were analyzed per condition. WT data from Figure 1 are presented alongside KO data for comparison. **: p < 0.0001; *: p = 0.0045, #: p = 0.03 by ANOVA, Tukey’s post hoc test).