KLHL3-Cab39 Disruption Drives WNK4-Independent Hyperkalemic Hypertension

Familial hyperkalemic hypertension, also known as pseudohypoaldosteronism type II (PHAII), is characterized by hypertension and hyperkalemic metabolic acidosis and involves dysregulation of the KLHL3 (Kelch-like 3)-cullin3 E3 ubiquitin ligase complex, affecting WNK1/4 (with-no-lysine kinase 1/4)-mediated SPAK (Ste20-related proline/alanine-rich kinase)/OSR1 (oxidative stress response kinase 1)-NCC (Na⁺-Cl^- cotransporter) signaling. How KLHL3 phosphorylation mutations modulate this pathway remains poorly defined.

We generated knock-in mouse models expressing phosphomimetic (S486D) or phosphodefective (S486G) mutations at Klhl3 S486 (homologous to human S433). Blood pressure, serum and urine biochemistries, immunoblotting, immunofluorescence, coimmunoprecipitation, in vitro ubiquitination assays, and molecular docking were performed. Klhl3 S486G mice were crossed with Wnk4^-/- mice to test WNK4 dependence.

Both knock-in strains exhibited typical PHAII features despite unchanged KLHL3 levels, with enhanced SPAK/OSR1-NCC phosphorylation, increased Cab39 levels, and unchanged WNK1/4 expression. Under high-salt loading, Klhl3 S486 knock-in mice maintained severe PHAII phenotypes, with persistently elevated Cab39 and NCC levels despite decreased WNK4 expression. Critically, Wnk4 deletion failed to rescue PHAII phenotypes in Klhl3 S486G mice, demonstrating that Klhl3 S486 mutations drive PHAII independently of WNK4. In vitro, human KLHL3 S433D/S433G mutations reduced KLHL3-Cab39 association and Cab39 ubiquitination, with a corresponding increase in Cab39 abundance. Deletion mapping identified KLHL3 residues 428 to 437 and Cab39 residues 314 to 324 as key determinants of the KLHL3-Cab39 association.

These findings indicate that the integrity of Ser486 itself, rather than its phosphorylation state, is required for KLHL3-mediated Cab39 ubiquitination and define a WNK4-independent PHAII mechanism driven by impaired Cab39 degradation and consequent SPAK/OSR1-Na+-Cl- cotransporter activation.