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Abstract: FR-PO617

Phosphor-Modified KLHL3 Knockin Mice Reveal WNK1/4-Independent SPAK/OSR1-NCC Activation in Pseudohypoaldosteronism Type II (PHAII)

Session Information

Category: Genetic Diseases of the Kidneys

  • 1202 Genetic Diseases of the Kidneys: Non-Cystic


  • Lin, Shih-Hua P., Tri-Service General Hospital Department of Internal Medicine, Taipei, Taiwan
  • Sung, Chih-Chien, Tri-Service General Hospital Department of Internal Medicine, Taipei, Taiwan
  • Cheng, Chih-Jen, Tri-Service General Hospital Department of Internal Medicine, Taipei, Taiwan

We have successfully created missense Klhl3M131V/+ knockin (KI) mice (human M78V mutation in BTB domain) and nonsense Klhl3W523X/+ KI mice (human W470X mutation in Kelch domain) to decipher the molecular mechanisms of PHAII. However, the phosphorylation site (S433) on KLHL3 regulated by several stimuli such as angiotensin II, insulin, and calcineurin inhibitors in PHAII have not been examined in vivo.


We generated and analyzed two missense Klhl3 KI mice with phosphor-modified at S486 site. The phenotypes of phosphomimetic Klhl3S486D/+ mice (human KLHL3 S433D mutation) and phosphodefective Klhl3S486G/+mice (human KLHL3 S433G mutation) were examined. The associated protein expression of their kidney tissue was evaluated by western blot and immunofluorescence.


Unlike the Wnks-dependent Spak/Osr1-Ncc activation in Klhl3M131V/+ and Klhl3W523X/+ KI mice, both Klhl3S486D/+ and Klhl3S486G/+mice recapitulating typical phenotypes of PHAII exhibited an enhanced phosphorylation of Spak/Osr1-Ncc but the “unchanged” Wnk1/4 and Klhl3 expression. Both phosphor-modified Klhl3 KI mice demonstrated a significantly increased expression of calcium binding protein 39 (Cab39) known to interact and stimulate Spak/Osr1, as compared to those in WT littermates, Klhl3M131V/+ and Klhl3W523X/+ mice. In vitro study showed that endogenous Cab39 interacted with KLHL3 and Cul3 complex, but not with WNK1/4. The simulation model demonstrated that KLHL3-WT and phosphor-modified KLHL3 mutants had different binding regions with Cab39.


Phosphor-modified KLHL3 KI mice exhibiting PHAII reveal a novel Wnks-independent Spak/Osr1-Ncc activation. Whether the phosphorylated status of KLHL3 S433 could affect its binding ability with Cab39 as a substrate for ubiquitination needs to be well validated.


  • Government Support – Non-U.S.