Abstract
ABSTRACTPseudohypoaldosteronism 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 Cul3G(−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.
Highlights
Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by hypertension, hyperkalemia, and metabolic acidosis (Gordon, 1986)
We demonstrated that Kelch-like family member 3 (KLHL3) bound to cullin 3 (Cul3) via its BTB domain and formed an E3 ligase for with-no-lysine kinase 4 (WNK4), and that PHAII-causing mutations in WNK4 and KLHL3 disrupted the formation of the WNK4–KLHL3–Cullin3 complex and impaired the ubiquitination and degradation of WNK4 (Mori et al, 2013; Ohta et al, 2013; Wakabayashi et al, 2013)
Generation of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene PHAII-causing mutations in Cul3 are known to cluster around exon 9 of the Cul3 gene, and some are located within the splice acceptor site in intron 8 (Boyden et al, 2012)
Summary
Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by hypertension, hyperkalemia, and metabolic acidosis (Gordon, 1986). Activation of the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubules is the main pathogenesis of this disease because all of its phenotypes are sensitive to thiazide treatment (Mayan et al, 2002). Mutations in with-nolysine kinase 1 (WNK1) and with-no-lysine kinase 4 (WNK4) were reported to be responsible for PHAII in 2001 (Wilson et al, 2001).
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