PMC

Schematic representation of the mechanism of the dominant negative effect caused by KLHL3 R528H.

Confirmation of the absence of KLHL3 expression and the presence of β-Gal expression by the knockout allele. Representative immunoblots of KLHL3 and β-Gal in the kidneys and brains of KLHL3^+/+, KLHL3^+/−, and KLHL3^−/− mice are shown, confirming the absence of the KLHL3 protein and expression of the β-Gal protein by the Klhl3 knockout allele.

The protein levels of WNK1 and WNK4 were higher in KLHL3^−/− mice than in KLHL3^R528H/+ mice. (A) Protein expression levels of WNK1 and WNK4 in kidneys from wild-type, KLHL3^−/−, and KLHL3^R528H/+ mice. The ability to degrade WNK kinases. This was conserved in KLHL3^R528H/+ mice but lost in KLHL3^−/− mice. (B) Densitometric analysis. Values are expressed as a ratio of the average signal in KLHL3^R528H/+ mice. n = 3 to 9; *, P < 0.05.

Increased salt sensitivity in KLHL3^−/− mice. (A) The blood pressure of KLHL3^+/+ and KLHL3^−/− mice after being fed a normal or a high-salt diet. (B) The observed blood pressure increased as a result of the high-salt diet. No significant difference in blood pressure was detected under a normal diet, whereas the increase of systolic blood pressure was significantly higher in KLHL3^−/− mice than in KLHL3^+/+ mice under a high-salt diet.

Increased WNK1 and WNK4 protein levels, and the activation of the WNK-OSR1/SPAK-NCC phosphorylation signaling cascade in the kidneys of KLHL3^−/− mice. (A) Representative immunoblots of WNK1 and WNK4 in the kidneys of KLHL3^+/+, KLHL3^+/−, and KLHL3^−/− mice. The expression levels of WNK1 and WNK4 and the phosphorylation of SPAK, OSR1, and NCC were significantly higher in the kidneys of KLHL3^−/− mice but not in KLHL3^+/− mice. (B) Densitometric analysis. Values are expressed as a ratio of the average signal in wild-type mice. n = 3 to 6; *, P < 0.05.

Generation of KLHL3 knockout/lacZ knock-in mice. (A) Targeting strategy for generating KLHL3 knockout/lacZ knock-in mice. The diagram shows the wild-type KLHL3 locus, the targeting vector, and the targeted locus before and after Cre recombination. The targeting vector was designed to delete exon 3 of KLHL3. In addition, this vector led to the expression of the lacZ gene under the control of the endogenous KLHL3 promoter. EN2-SA, Engrailed-2 splice acceptor. (B) Verification of homologous recombination and genotyping PCR of the genomic DNA of the mice.

WNK kinases only increased in the kidneys of KLHL3^−/− mice. (A) Representative immunoblots of β-Gal and WNK1, WNK3, and WNK4 in KLHL3-expressing tissues in KLHL3^+/+ and KLHL3^−/− mice. In KLHL3^−/− mice, the expression levels of WNK1, WNK3, and WNK4 did not increase in any organ other than the kidney, where WNK1 and WNK4 increased. N.D., not detected. (B) Immunoblotting of WNK1 and WNK3 protein levels in hippocampus and cortex from KLHL3^−/− mice. No significant differences in WNK protein levels were detected between wild-type and KLHL3^−/− mice. We repeated the same experiments three times with consistent results.

Dominant negative effect of KLHL3 R528H requires dimer formation of KLHL3. (A) FLAG-tagged KLHL3 was coimmunoprecipitated with Halo-tagged KLHL3 and Halo-tagged R528H KLHL3 in HEK293T cells. Wild-type KLHL3 could form a homodimer and heterodimer with wild-type and mutant KLHL3 R528H, respectively. IP, immunoprecipitation. (B, upper panel) Coexpression experiments of KLHL3 R528H and wild-type KLHL3. Compared to the cells transfected with wild-type KLHL3 alone, the degradation of WNK4 protein was significantly decreased by the addition of KLHL3 R528H. In addition, this effect was cancelled when the BTB-BACK domain, containing the binding site for dimer formation, was deleted from the mutant KLHL3 R528H, suggesting that the dominant negative effect of mutant KLHL3 R528H required dimer formation of KLHL3. (B, lower panel) Densitometry analysis of avidin binding to Bio-ease-WNK4.

Tissue and intratissue distribution of KLHL3 was confirmed by an immunoblot of β-Gal and LacZ staining. (A) Immunoblotting to detect the β-Gal protein level in each organ from KLHL3 knockout/lacZ knock-in mice. Representative immunoblots of β-Gal in each of the organs of wild-type and KLHL3 homozygous knockout/lacZ knock-in mice are shown. β-Gal was strongly detected in the kidney and brain and weakly detected in the eye, testis, lung, heart, liver, stomach, and colon. (B) LacZ staining of the kidney and brain in KLHL3 knockout/lacZ knock-in mice. Strong LacZ staining was observed in the hippocampus and cortex of the brain and in the distal convoluted tubules of the kidney. (C) Double immunofluorescence of β-Gal and the Na-Cl cotransporter (NCC) in the kidneys of wild-type and KLHL3 knockout/lacZ knock-in mice. The β-Gal signal colocalized with NCC, indicating that KLHL3 was present in the distal convoluted tubules. Scale bars, 10 μm.