背景:研究报道了海带样蛋白3(KLHL3)-Cullin3(CUL3)E3连接酶被无赖氨酸激酶4(WNK4)泛素化。受损的WNK4泛素化在家族性高钾性高血压中起关键作用(FHHt,也称为假醛固酮增多症II型),由噻嗪类敏感的氯化钠共转运(NCC)的过度作用引起。此外,研究人员还发现,饮食中的钾缺乏会激活沿肾远曲小管(DCT)的NCC。然而,钾与WNK4之间的关系尚不清楚。
方法:在本研究中,我们进行了体外和体内实验,以证实KLHL3依赖的WNK4降解通过Neddylation和自噬途径受到钾的影响。体外,将WNK4和KLHL3质粒用脂转染胺2000共转染到HEK293细胞系中,然后与不同浓度的钾(1mmol/L和10mmol/L)孵育24h,并进一步用MLN4924或自噬抑制剂或MLN4924和自噬抑制剂两者分别处理另外24小时。在体内,我们创建了用低钾或高钾饮食喂养的小鼠,然后在实验组中注射MLN4924.WNK4,pWNK4,KLHL3,NEDD8,LC3,在体外和体内实验中通过蛋白质印迹法检测P62。
结果:我们发现,当在体外和体内抑制neddylation时,WNK4的丰度和磷酸化增加。此外,在低钾(LK)组中,pWNK4,WNK4,NEDD8和KLHL3的丰度增加。抑制自噬可以在一定程度上改善钾对WNK4丰度和活性的影响。
结论:这些发现提示了钾在WNK4降解过程中的复杂调控。低钾可以激活WNK4,这可能与内化和自噬有关,但其机制有待进一步研究。
BACKGROUND: Studies reported that kelch-like protein 3 (KLHL3)-Cullin3(CUL3) E3 ligase ubiquitinated with-no-lysine kinase 4 (
WNK4). Impaired
WNK4 ubiquitination plays a key role in Familial hyperkalemic hypertension (FHHt, also called pseudohypoaldosteronism type II) which results from overaction of thiazide-sensitive sodium chloride cotransport (NCC). In addition, researchers have also found that dietary potassium deficiency activates NCC along the renal distal convoluted tubule (DCT). However, the underlying mechanism remains unclear about the relationship between potassium and
WNK4.
METHODS: In the present study, we conducted in vitro and in vivo experiments to confirm that KLHL3-dependent
WNK4 degradation is affected by potassium through the neddylation and autophagy pathway. In vitro, the
WNK4 and KLHL3 plasmids were cotransfected into HEK293 cell lines by lipofectamine 2000, and then incubated with different potassium concentrations (1mmol/L and 10mmol/L) for 24 h, and further treated with MLN4924 or the autophagy inhibitor or both of MLN4924 and the autophagy inhibitor for another 24 h respectively. In vivo, we created mice that were fed with low or high potassium diets and then were injected MLN4924 in the experimental groups. The expression of WNK4, pWNK4, KLHL3, NEDD8, LC3 ,and P62 was detected by western blotting in vitro and vivo experiments.
RESULTS: We found that the abundance and phosphorylation of WNK4 increase when neddylation is inhibited both in vitro and vivo. Furthermore, the abundance of pWNK4, WNK4, NEDD8, and KLHL3 was increased in the low potassium (LK) group. Inhibiting autophagy can ameliorate the effect of potassium on the abundance and activity of WNK4 to some extent.
CONCLUSIONS: These findings suggest a complex regulation of potassium in the degradation of WNK4. Low potassium can activate WNK4, which may be related to neddylation and autophagy, but the mechanism needs to be further studied.