KS-WNK1 Is Necessary to Deactivate the Sodium-Chloride Cotransporter (NCC) Under NCC-Suppressive Conditions

Objective: The sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays a key role in potassium (K + ) balance and blood pressure regulation. NCC activity is regulated by With-No-Lysine (WNK) kinases, particularly WNK4, which activates the STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC signaling pathway. Kidney-specific WNK1 (KS-WNK1), a WNK1 isoform is expressed in the DCT. Our prior studies suggest that KS-WNK1 promotes WNK4 degradation, and its absence results in increased NCC activation. Based on these findings, we investigated whether the absence of KS-WNK1 prevents NCC deactivation under NCC-suppressive conditions. Hypothesis: KS-WNK1 facilitates NCC deactivation under NCC-suppressive conditions by promoting WNK4 degradation or destabilization. Methods: To test this hypothesis, we used mice with a DCT-specific knockout (KO) of KS-WNK1 to assess its role in NCC regulation. Protein levels were analyzed by western blotting, and gene expression was measured by quantitative PCR. Mice were fed high salt and potassium diets designed to elevate blood potassium levels within physiological limits while maintaining high salt intake. This protocol aimed to induce a physiological robust NCC-suppressive environment to assess the effects of KS-WNK1 absence on NCC activity. Results: The NCC-deactivating diet significantly reduced phosphorylated NCC (pNCC) (a surrogate for NCC activity) levels in both wild-type and KS-WNK1 KO mice. However, KS-WNK1 KO mice exhibited a less pronounced reduction in pNCC, resulting in higher pNCC levels compared to wild-type mice on the same diet. Additionally, under the NCC-deactivating diet, KS-WNK1 KO mice showed increased WNK4 protein and phosphorylated SPAK levels, despite a reduction in WNK4 mRNA expression. These results suggest that KS-WNK1 regulates WNK4 stability post-translationally rather than through transcriptional control. Thus, KS-WNK1-mediated WNK4 degradation is essential for NCC deactivation. Conclusion: KS-WNK1 is necessary for NCC deactivation under NCC-suppressive conditions by promoting WNK4 degradation. The absence of KS-WNK1 leads to sustained NCC activity, highlighting its critical role in regulating the WNK4-SPAK-NCC pathway. These findings provide new insights into the molecular mechanisms of KS-WMK1 mediated electrolyte balance and may suggest potential therapeutic targets for hypertension and related disorders. DK093501 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.