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Published on: December 2, 2014
Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3
Yuya Araki1, Tatemitsu Rai2, Eisei Sohara1
1Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-0034, Japan.
Abstract:
Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207-1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3(G(-1)A/+) knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes.
Insights
Pseudohypoaldosteronism type II (PHAII) is a rare genetic hypertension. Cul3 mutations causing PHAII do not lead to exon skipping in kidneys, suggesting other mechanisms are involved in this kidney-specific disease.
Area of Science:
- Nephrology
- Genetics
- Molecular Biology
Background:
- Pseudohypoaldosteronism type II (PHAII) is an inherited hypertension linked to mutations in WNK, KLHL3, and Cul3 genes.
- The Cul3-KLHL3 E3 ligase complex normally ubiquitinates WNK4, reducing its expression; PHAII mutations disrupt this process.
- The specific role of Cul3 mutations in PHAII pathogenesis, particularly in the kidneys, remains unclear.
Purpose of the Study:
- To investigate the in vivo molecular mechanisms of PHAII caused by Cul3 mutations in the kidney.
- To determine if Cul3 mutations lead to exon skipping in the kidney and contribute to PHAII phenotypes.
Main Methods:
- Generated knock-in mice with a PHAII-associated Cul3 mutation (c.1207-1G>A).
- Analyzed Cul3 mRNA levels and exon 9 skipping in the kidneys of heterozygous and homozygous knock-in mice.
- Assessed PHAII phenotypes in heterozygous knock-in mice.
Main Results:
- Heterozygous Cul3(G(-1)A/+) mice did not display PHAII phenotypes or exon 9 skipping in kidneys.
- Cul3 mRNA levels were halved in heterozygous knock-in mouse kidneys.
- Homozygous knock-in mice were nonviable, indicating the mutant allele acts like a knockout.
Conclusions:
- Cul3 mutations causing PHAII do not induce exon 9 skipping in mouse kidneys.
- Reduced Cul3 expression alone is insufficient to cause PHAII.
- The pathogenic role of mutant Cul3 protein, rather than exon skipping, likely underlies kidney-predominant PHAII phenotypes.
