Susa Koichiro, Sohara Eisei, Takahashi Daiei, Okado Tomokazu, Rai Tatemitsu, Uchida Shinichi
Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo, Tokyo, 113-8519, Japan.
Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo, Tokyo, 113-8519, Japan.
Biochem Biophys Res Commun. 2017 Sep 23;491(3):727-732. doi: 10.1016/j.bbrc.2017.07.121. Epub 2017 Jul 22.
WNK-OSR1/SPAK-NCC signaling cascade is important for regulating salt balance and blood pressure. Activation of WNK-OSR1/SPAK-NaCl cotransporter (NCC) cascade increases sodium reabsorption in the kidney, leading to pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. It has been previously demonstrated that the amount of phosphorylated and total NCC markedly decreased in WNK4 mice, indicating that WNK4 plays a major role for activation of OSR1/SPAK-NCC signaling. However, it is unclear whether absence of WNK4 can be compensated by other WNK kinases. We recently reported that KLHL3 knock-in mice, a PHAII model, exhibited augmented activation of OSR1/SPAK-NCC signaling by increased protein levels of both WNK1 and WNK4 due to impaired protein degradation by the mutant KLHL3. In this study, we sought to determine the contribution of WNK4 to OSR1/SPAK-NCC signaling using an in vivo model which shows extremely increased WNK1 with absence of WNK4. We generated WNK4KLHL3 mice and WNK4KLHL3 mice by crossing WNK4 mice with KLHL3 mice. Thereafter, WNK-OSR1/SPAK-NCC phosphorylation signal cascade was examined in kidneys from these mice. As expected, both WNK4KLHL3 mice and WNK4KLHL3 mice demonstrated increased WNK1 in the kidney, due to the KLHL3 mutation, and WNK4 deficiency. However, phosphorylation of SPAK and NCC at distal convoluted tubules were almost completely absent even in WNK4KLHL3 mice. In conclusion, increased WNK1 was unable to compensate for WNK4 deficiency and phosphorylate the NCC, indicating that WNK4 is indispensable for the onset of PHAII.
WNK-OSR1/SPAK-NCC信号级联对于调节盐平衡和血压至关重要。WNK-OSR1/SPAK-氯化钠协同转运蛋白（NCC）级联的激活会增加肾脏中的钠重吸收，导致以盐敏感性高血压和高钾血症为特征的II型假性醛固酮减少症（PHA II）。先前已经证明，WNK4基因敲除小鼠中磷酸化NCC和总NCC的量显著减少，这表明WNK4在激活OSR1/SPAK-NCC信号中起主要作用。然而，尚不清楚WNK4的缺失是否能被其他WNK激酶代偿。我们最近报道，PHAII模型KLHL3基因敲入小鼠由于突变的KLHL3导致蛋白质降解受损，WNK1和WNK4的蛋白质水平增加，从而表现出OSR1/SPAK-NCC信号的增强激活。在本研究中，我们试图使用一种体内模型来确定WNK4对OSR1/SPAK-NCC信号的作用，该模型显示WNK1极度增加而WNK4缺失。我们通过将WNK4基因敲除小鼠与KLHL3基因敲除小鼠杂交，培育出了WNK4KLHL3小鼠和WNK4KLHL3小鼠。此后，检测了这些小鼠肾脏中的WNK-OSR1/SPAK-NCC磷酸化信号级联。正如预期的那样，由于KLHL3突变和WNK4缺陷，WNK4KLHL3小鼠和WNK4KLHL3小鼠的肾脏中WNK1均增加。然而，即使在WNK4KLHL3小鼠中，远端曲管中SPAK和NCC的磷酸化也几乎完全缺失。总之，WNK1增加无法代偿WNK4缺陷并使NCC磷酸化，这表明WNK4对于PHAII的发病是不可或缺的。
