대한신장학회

논문분류	춘계학술대회 초록집
제목	Elucidating the Function of WNKs on Pseudohypoaldosteronism Type II Caused by KLHL3 BTB Domain Mutation
저자	Chien-Ming Lin1, Chih-Jen Cheng2, Sung-Sen Yang2, Shih-Hua Lin2
출판정보	2018; 2018(1):
키워드	Cullin 3 \| Gitelman syndrome \| Kelch-like 3 \| Pseudohypoaldosteronism type II \| With-no-lysine (WNK) kinases
초록	Objectives: Enhanced SPAK/OSR1-NCC cascade caused by mutations in Kelch-like 3 (KLHL3) or Cullin3 (Cul3) involved in WNK1/4 ubiquitination is known to cause pseudohypoaldosteronism type II (PHAII). It remains unclear that which WNK kinases is the major regulator in KLHL3 mutation-causing PHAII. Methods: We generated and analyzed Klhl3 knock-in (KI) mice carrying a missense M131V mutation in the BTB domain (corresponding to human KLHL3 M78V mutation) and further crossed them with Wnk4 -/- mice generated by targeting disruption from the promoter to exon 2 of Wnk4. The molecular mechanisms of PHAII regulated by KLHL3 BTB domain mutation were further evaluated by western blot, immunogold labelling, qRT-PCR in microdissected renal tubules, and co-immunoprecipitation (Co-IP). Results: Klhl3M131V/+ KI mice exhibited typical feature of PHAII with hypertension with suppressed PRA and hyperkalemic metabolic acidosis. Their kidney tissues showed an unchanged KLHL3, decreased Cul3, and increased WNK1/4 expression along with an enhanced downstream SPAK/OSR1-N(K)CC phosphorylation. Their Cul3 protein expression in the cytosol of distal convoluted tubules cells was significantly attenuated on immunogold labelling electron microscopy. In microdissected renal tubules, Klhl3M131V/+ KI mice expressed high levels of Wnk4 mRNA in the distal nephron. In vitro Co-IP showed the KLHL3 BTB domain mutation retained intact interaction with WNKs but reduced binding to Cul3, thus leading to the increased abundance of total WNKs. KlhlM131V/M131V．Wnk4 -/- double transgenic mice still showed the overwhelming phenotypes of Gitelman syndrome (GS) with an increased expression of WNK1 but a decreased phosphorylation of SPAK and NCC. Conclusions: Klhl3M131V/+ KI mice feature typical PHAII with a simultaneous increase of WNK1 and WNK4 through the impaired KLHL3 BTB domain binding to Cul3 and fail to correct the GS-like phenotypes of the Wnk4 -/- mice. WNK4 is a vital WNKs in regulating SPAK/OSR1-N(K)CC signalling cascade under the circumstances of KLHL3 BTB domain mutation. Generation of Klhl3M131V/+ KI mice
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