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. 2018 Mar 30;29(5):1449–1461. doi: 10.1681/ASN.2017101091

Figure 5.

Figure 5.

Mouse protein 25 (Mo25) stimulates Fray activity in vitro, and it is required for tubule transepithelial transport in stimulated (hypotonic) conditions. (A) Mo25 stimulates autophosphorylation of Fray (Drosophila Ste20-related proline/alanine-rich kinase [SPAK]/oxidative stress response 1 [OSR1] homolog; upper two bands) as well as sodium-potassium 2 chloride cotransporter (NKCC) phosphorylation (lower band) in vitro. The effect is more pronounced with FrayT206E, in which the T-loop Thr that is the target of With No Lysine kinase (WNK) phosphorylation is mutated to a phosphomimicking Glu. In vitro kinase assays were performed with purified Drosophila Fray, D. melanogaster mouse protein 25 (DmMo25), and the N terminus of Drosophila NKCC, Ncc69 (amino acids 1–204). A representative gel is shown of three independent experiments (Supplemental Figure 5). WT, wild type. (B) DmMo25 is required in the tubule principal cell for hypotonicity-stimulated transepithelial potassium flux. Transepithelial potassium flux was measured in control tubules (w; c42-GAL4/+) and DmMo25 knockdown tubules (w; UAS-DmMo25RNA interference (RNAi)/+; c42-GAL4/+) bathed in isotonic standard bathing medium or hypotonic medium (standard bathing medium diluted with water). Two-way ANOVA revealed significant effects of genotype (P<0.001), condition (P<0.001), and interaction (P<0.01). In a Sidak multiple comparisons test, hypotonic conditions resulted in a significant increase in potassium flux in control tubules but no change in DmMo25 knockdown tubules (P=0.82). There was no significant difference between control and DmMo25 knockdown tubules in isotonic conditions (P=0.67), whereas a significant difference was observed in hypotonic conditions (P<0.001). ****Adjusted P value <0.001. (C) Expression of mammalian Mo25 rescues the decreased potassium flux phenotype of DmMo25 knockdown tubules. Transepithelial potassium flux was measured in hypotonic conditions in control tubules (w; c42-GAL4/+), DmMo25 principal cell knockdown tubules (w; UAS-DmMo25RNAi/+; c42-GAL4/+), and DmMo25 knockdown tubules expressing mouse Mo25 (w; UAS-DmMo25RNAi/+; UAS-MmMo25/c42-GAL4). One-way ANOVA (P<0.001). In a Tukey multiple comparison test comparing all genotypes with one another, adjusted P values were <0.001 for the difference between w; c42-GAL4/+ and w; UAS-DmMo25RNAi/+; c42-GAL4/+ and also for the difference between w; UAS-DmMo25RNAi/+; c42-GAL4/+ and w; UAS-DmMo25RNAi/+; UAS-MmMo25/c42. Adjusted P value was 0.04 for the difference between w; c42-GAL4/+ and w; UAS-DmMo25RNAi/+; UAS-MmMo25/c42-GAL4. ****Adjusted P value <0.001.