NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells
NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells
复制标题
DOI：
10.1111/boc.201400069
复制
发表时间：
2015-04-01
影响因子：
2.7
通讯作者：
Svelto, Maria
中科院分区：
文献类型：
--
作者：
Carmosino, Monica;Gerbino, Andrea;Svelto, Maria
背景信息钙敏感受体（CaR）的功能获得性A843 E突变导致Bartter综合征5型。携带这种CaR变体的患者显示在亨利氏袢的粗升支（TAL）中肾NaCl重吸收显著降低，导致在肾Na+和Cl-离子转运蛋白中不存在突变的情况下肾NaCl损失。这种临床表型的分子机制还不完全清楚。我们调查，在人胚肾293（HEK 293）细胞和猪肾上皮（LLC-PK 1）细胞，功能串扰的CaR-A843 E与Na+：K+：2Cl（-）的协同转运，NKCC 2，它提供了NaCl重吸收在TAL.ResultsThe表达的CaR突变体没有改变顶端定位的NKCC 2在LLC-PK 1细胞。然而，与对照野生型CaR（CaR WT）转染细胞相比，CaR-A843 E转染细胞的稳态NKCC 2磷酸化和活性降低。值得注意的是，低Cl-依赖性NKCC 2激活也强烈抑制表达的CaR-A843 E突变体。P450羟化酶（CYP 4）或磷脂酶A2（PLA 2）阻滞剂的使用表明，这种作用可能是由花生四烯酸（AA）代谢物介导的。结论数据表明，激活的CaR影响调节NKCC 2活性的细胞内途径，而不是NKCC 2在肾细胞中的细胞内运输，并进一步阐明在Bartter综合征5型中活性CaR突变体所起的病理作用。
Background informationThe gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na+ and Cl- ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na+:K+:2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL.ResultsThe expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl--dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 -hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites.ConclusionsThe data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5.