Cab39 deletion overrides the WNK4–SPAK stimulatory effect via SPAK sequestration in non-WNK body puncta

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) regulates renal sodium reabsorption and blood pressure. NCC activity is governed by the WNK–SPAK signaling cascade, spatially organized by scaffolding proteins. KS-WNK1 is essential for WNK body formation, and its deletion enhances NCC activity by increasing WNK abundance. Cab39 stabilizes SPAK and promotes catalytic function, and its deletion suppresses NCC activity by restricting SPAK in WNK bodies. These opposing effects raise a central question: how do scaffolds coordinate kinase localization and function to regulate NCC? Although SPAK-containing puncta are often interpreted as WNK bodies, it remains unclear whether all puncta share the same composition or function. Therefore, the objective of this study is twofold: To determine the combined effect on NCC activity when Cab39, which suppresses NCC, and KS-WNK1, which enhances NCC, are both deleted. To assess whether Cab39 deletion in the absence of KS-WNK1 can still drive SPAK puncta formation, despite KS-WNK1 being required for canonical WNK body assembly. We hypothesize that Cab39 deletion overrides the NCC-activating effect of KS-WNK1 deletion by sequestering SPAK into distinct puncta. We generated mice with DCT-specific deletion of both Cab39 isoforms and KS-WNK1 (TKO), fed control, low-potassium, or high-potassium diets for 4–5 days. NCC, SPAK, and WNK4 expressions and localization were assessed by immunoblotting, immunofluorescence, and qPCR. WNK4 expression was elevated in TKO, (mean ± SEM): control, 100 ± 0.55; Cab39 isoforms double knockout (DKO), 85.10 ± 2.21; TKO, 136.9 ± 5.95. Statistical comparisons: control vs. DKO, p = 0.06; control vs. TKO, *p = 0.001; DKO vs. TKO, *p = 0.0001 (*p < 0.05). Despite increased WNK4 and preserved SPAK signal, TKO mice exhibited marked NCC hypofunction, as indicated by phosphorylated NCC (pNCC, a surrogate for NCC activity) levels, (mean ± SEM): control, 100 ± 17.44; DKO, 0.463 ± 0.463; TKO, 0.00 ± 0.00. Statistical comparisons: control vs. DKO, *p = 0.001; control vs. TKO, *p = 0.001; DKO vs. TKO, p = 0.999 (*p < 0.05). SPAK accumulated in cytoplasmic puncta resembling WNK bodies but lacking KS-WNK1, confirming structural distinction. Although only a subset of puncta exhibited co-localization with WNK4, these structures were enriched for phosphorylated SPAK, indicating that WNK4-dependent phosphorylation and activation of SPAK precede and likely drive puncta formation. Under high-potassium conditions, puncta were absent, indicating dependence on upstream kinase activity. Thus, Cab39 is essential for NCC regulation by SPAK, ensuring proper kinase localization. Its deletion causes SPAK to accumulate in puncta even without KS-WNK1, suggesting formation of structurally distinct condensates with potentially different composition, regulation, function, and fate. This work is supported by NIH grant R01DK093501 to E.D. and P.A.W. This abstract was presented at the American Physiology Summit 2026 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.