Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene

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.

Keywords: Cullin 3; Hypertension; PHAII.

Fig. 1.

Sequence homology between human and mouse cullin 3. (A) Exon–intron structure of mouse cullin 3 (Cul3; NCBI Reference Sequence: NC_000067.6). Boxes represent exons. (B) Comparison of the sequence homology between mouse Cul3 (NCBI Reference Sequence: NC_000067.6) and human Cul3 (NCBI Reference Sequence: NG_032169.1). The sequence of Cul3 is highly conserved between the species; indeed, the length of exon 9 is 171 base pairs in both species. The indicated mutations are pseudohypoaldosteronism Type II-causing mutations in Cul3 located within the splice acceptor site in intron 8.

Fig. 2.

Targeting strategy for generating cullin 3 knock-in mice. (A) Wild-type cullin 3 (Cul3) locus, the targeting construct, and the targeted locus before and after FLPe recombination. Primers F1, F2, F3, R1, R2, and R3 are shown on the targeted locus. Probes used for southern blotting are shown as 5′ and 3′ probes. (B) Verification of homologous recombination by southern blotting. Genomic DNA was digested with EcoRI, and southern blotting was performed with the 5′ probe. The 6.6-kbp band was generated from the wild-type allele and the 4.7-kbp band was generated from the mutant allele (top). Genomic DNA was digested with EcoRV, and southern blotting was performed with the 3′ probe. The 13.6-kbp band was generated from the wild-type allele and the 9.4-kbp band was generated from the mutant allele (bottom). (C) Polymerase chain reaction (PCR)-verification of homologous recombination using the genomic DNA of selected embryonic stem cell (ESC). The locations for primers F1, F2, R1, and R2 are shown in A. The 2.95-kbp (5′-side PCR) and 9.2-kbp (3′-side PCR) bands were generated from the mutant allele. Primer sets were designed to prevent amplification of the wild-type Cul3 gene. WT: host ES cells. (D) Direct sequencing of the PCR product covering the mutation site. WT: wild type. G(−1)A: Cul3 c.1207 −1G>A. T(−6)G: Cul3 c.1207 −6T>G.

Fig. 3.

Reverse-transcription polymerase chain reaction of the spliced RNA. Reverse-transcription (RT) polymerase chain reaction (PCR) of the spliced RNA was performed. (A,B) PCR using a primer set (F4 and R4) flanking exon 9. Wild-type Cullin 3 (Cul3) cDNA produced a single product that included exon 9 (664 bp). If exon 9 of CUL3 was skipped, the expected PCR product would be 493 bp. We detected only the bands including exon 9. (C) Representative RT-PCR sequences. Complementary DNA with properly spliced junctions between exons 8 and 9 was confirmed in both types of knock-in mice. WT: wild type. G(−1)A: Cul3 c.1207 −1G>A knock-in mice. T(−6)G: Cul3 c.1207 −6T>G knock-in mice.

Fig. 4.

Expression levels of cullin 3 protein and mRNA and proteins of the WNK–OSR1/SPAK–NCC phosphorylation signaling cascade in the kidneys of Cullin 3^G(−1)A/+ knock-in mice. (A) Immunoblots of proteins of the WNK–OSR1/SPAK–NCC signaling cascade in the kidneys of wild-type (WT) and cullin 3 (Cul3)^G(−1)A/+ heterozygous knock-in mice. (B) Densitometry analysis. Values are expressed as a ratio of the average signal in WT mice. Cul3 expression levels in Cul3^G(−1)A/+ heterozygous knock-in mice were approximately half that of WT mice. There were no significant differences in proteins expression levels of the WNK–OSR1/SPAK–NCC phosphorylation signaling cascade between Cul3^G(−1)A/+ heterozygous knock-in and WT mice. (C) Quantitative polymerase chain reaction (PCR) analysis of Cul3 mRNA levels. SYBR Green quantitative PCR was used to quantify mRNA levels in the kidneys of WT mice (n=7) and Cul3^G(−1)A/+ mice (n=7). WT: wild type. G(−1)A: Cul3^G(−1)A/+. *P<0.05 compared with wild-type mice, data presented as mean±s.e.m.

Fig. 5.

Expression levels of cullin 3 protein and mRNA and proteins of the WNK-OSR1/SPAK-NCC phosphorylation signaling cascade in the kidneys of cullin 3^{T(−6)G/T(−6)G} knock-in mice. (A) Immunoblots of proteins of the WNK–OSR1/SPAK–NCC signaling cascade in the kidneys of wild-type (WT) and cullin 3 (Cul3) ^{T(−6)G/T(−6)G} homozygous knock-in mice. (B) Densitometry analysis. Values are expressed as the ratio of the average signal in WT mice. The level of Cul3 protein expression in Cul3^{T(−6)G/T(−6)G} mice was three-quarters that of WT mice. There were no significant differences in proteins expression levels of the WNK–OSR1/SPAK–NCC phosphorylation signaling cascade between Cul3^{T(−6)G/T(−6)G} and WT mice. (C) Quantitative polymerase chain reaction (PCR) analysis of Cul3 mRNA levels. SYBR Green quantitative PCR was used to quantify mRNA levels in the kidneys of WT mice (n=7) and Cul3^{T(−6)G/T(−6)G} mice (n=9). WT: wild-type mice. T(−6)G: Cul3^{T(−6)G/T(−6)G} mice. *P<0.05 compared with WT mice, data presented as mean±s.e.m.

Fig. 6.

Blood pressure and heart rate in wild-type, cullin 3^G(−1)A/+ and cullin 3^{T(−6)G/T(−6)G} mice. No significant differences in blood pressures or heart rate were evident between WT and cullin 3 (Cul3)^G(−1)A/+ mice or WT and Cul3^{T(−6)G/T(−6)G} mice. WT: wild type mice. G(−1)A: Cul3^G(−1)A/+ mice. T(−6)G: Cul3^{T(−6)G/T(−6)G} mice. Data presented as mean±s.e.m.