Division of Nephrology, Department of Medicine, Tri-Service General Hospital, Taipei, Taiwan;
J Am Soc Nephrol. 2013 Oct;24(10):1587-97. doi: 10.1681/ASN.2012070742. Epub 2013 Jul 5.
A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.
噻嗪类敏感钠氯共转运蛋白（NCC）中的 T60M 突变在 Gitelman 综合征（GS）患者中很常见。该突变阻止了 Ste20 相关脯氨酸和丙氨酸丰富激酶（SPAK）/氧化应激反应激酶-1（OSR1）介导的 NCC 磷酸化，并改变了体外 NCC 转运体活性。在这里，我们使用新型 Ncc T58M（人 T60M）敲入小鼠模型研究了 NCC 磷酸化在体内的生理效应。Ncc(T58M/T58M) 小鼠表现出 GS 的典型特征，对噻嗪类利尿剂的反应减弱。尽管表达正常水平的 Ncc mRNA，但这些小鼠的总 Ncc 和 p-Ncc 蛋白水平较低，低盐饮食增加 p-Spak 后也不会改变。与定位于远端卷曲小管细胞顶膜的野生型 Ncc 不同，T58M Ncc 主要定位于细胞质区域，并导致晚期远端卷曲小管体积增加。在 MDCK 细胞中，外源性表达磷酸化缺陷的 NCC 突变体减少了总蛋白表达水平和膜稳定性。此外，我们的分析发现，具有纯合 NCC T60M 突变的 GS 患者的总尿 NCC 排泄量减少。当 Wnk4(D561A/+) 小鼠（一种表达激活的 Spak/Osr1-Ncc 的假性醛固酮增多症 II 型模型）与 Ncc(T58M/T58M) 小鼠杂交时，总 Ncc 和 p-Ncc 蛋白水平降低，GS 表型持续存在，超过了高血压表型。总体而言，这些数据表明，SPAK 介导的 NCC 在 T60 处的磷酸化调节 NCC 的稳定性和功能，并且该残基的缺陷磷酸化纠正了假性醛固酮增多症 II 型的表型。
