Kidney Week

Abstract: FR-PO593

Generation and Analysis of Pseudohypoaldosteronism Type II Knockin Mice Caused by Nonsense Mutation in the Kelch Domain of KLHL3

Session Information

• Fluid and Electrolytes: Basic - I
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Lin, Chien-Ming, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan

Chien-Ming Lin,

• Yang, Sung-Sen, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan

Sung-Sen Yang,

• Lin, Shih-Hua P., Tri-Service General Hospital, Neihu, Taiwan

Shih-Hua P. Lin,

Background

Mutations in Kelch-like 3 (KLHL3) are the most common causative genes detected in patients with pseudohypoaldosteronism type II (PHAII). Although Klhl3^R528H/+ knock-in (KI) mice carrying a missense mutation in kelch repeat domain has been reported, the nonsense KLHL3 mutation-causing PHAII in the same domain have not been fully investigated in vivo.

Methods

We generated and analyzed mutant Klhl3 KI mice harboring a nonsense W523X mutation in the kelch domain (corresponding to human KLHL3 W470X mutation). The associated protein expression of their kidney tissue was evaluated by western blot and immunofluorescence. The co-immunoprecipitation (co-IP) method was conducted to clarify the role of KLHL3 kelch repeat domain.

Results

Both heterozygous and homozygous Klhl3^W523X/+ KI mice exhibited a significantly elevated systolic blood pressure (p < 0.05), secondary hyporeninemia (p < 0.05), a higher serum potassium level (K⁺) (p < 0.05) with decreased fractional excretion of K⁺ (FE_K; p < 0.05) and a higher serum chloride level but lower bicarbonate level (p < 0.05). Their kidney tissues showed decreased levels of KLHL3 protein along with an enhanced downstream WNK1/4-SPAK/OSR1-N(K)CC phosphorylation. In vitro, co-IP demonstrated human KLHL3 harboring the PHAII-causing W470X mutation resulted in a decreased expression of total KLHL3 protein with truncated KLHL3 protein, leading to impaired binding affinity of KLHL3 to WNK1/4.

Conclusion

Klhl3^W523X/+ KI mice feature typical PHAII with a simultaneous increase of WNK1 and WNK4 through impaired binding of the KLHL3 kelch domain to WNKs.