Department of Physiology, University of Tübingen, Germany.
Am J Physiol Cell Physiol. 2012 Aug 15;303(4):C416-26. doi: 10.1152/ajpcell.00420.2011. Epub 2012 May 30.
The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.
氧化应激反应激酶 1（OSR1）被 WNK（无激酶）激活，反过来又刺激噻嗪类敏感的 Na-Cl 共转运蛋白（NCC）和速尿敏感的 Na-K-2Cl 共转运蛋白（NKCC），从而有助于运输和细胞体积调节。关于 OSR1 的肾脏外功能知之甚少。本研究分析了 OSR1 活性降低对树突状细胞（DCs）功能的影响，DCs 是连接先天和适应性免疫的抗原呈递细胞。从杂合 WNK 抗性 OSR1 敲入小鼠（osr(KI)）和野生型小鼠（osr(WT)）的骨髓中培养 DCs。通过流式细胞术分析中的前向散射估计细胞体积，通过 2'，7'-二氯二氢荧光素二乙酸酯荧光估计 ROS 产生，通过 2'，7'-双（2-羧乙基）-5-（和-6）-羧基荧光素荧光估计细胞内 pH 值（pH(i)），通过铵脉冲后的 Na(+)-依赖性再碱化和利用 Transwell 室的迁移来估计 Na(+)/H(+)交换器活性。DCs 表达 WNK1、WNK3、NCC、NKCC1 和 OSR1。osr(KI) DCs 中的磷酸化 NKCC1 减少。osr(KI) 和 osr(WT) DCs 中的细胞体积和 pH(i)相似，但 osr(KI) DCs 中的 Na(+)/H(+)交换器活性和 ROS 产生高于 osr(WT) DCs。在 LPS 处理之前，osr(KI) 和 osr(WT) DCs 的迁移相似。然而，LPS（1μg/ml）增加了 osr(WT) DCs 的迁移，但不增加 osr(KI) DCs 的迁移。Na(+)/H(+)交换器 1 抑制剂 cariporide（10μM）使 osr(KI) DCs 的细胞体积、细胞内活性氧（ROS）形成、Na(+)/H(+)交换器活性和 pH(i)降低的程度大于 osr(WT) DCs。LPS 增加了 osr(WT) DCs 的细胞体积、Na(+)/H(+)交换器活性和 ROS 形成，但不增加 osr(KI) DCs 的细胞体积、Na(+)/H(+)交换器活性和 ROS 形成，并使 osr(KI) 和 osr(WT) DCs 之间的差异减弱。osr(WT) DCs 中的 NKCC1 抑制剂呋塞米（100 nM）增加 Na(+)/H(+)交换器活性，使其与 osr(KI) DCs 相似。氧化应激（10μM 叔丁基过氧化物）增加了 osr(WT) DCs 的 Na(+)/H(+)交换器活性，但不增加 osr(KI) DCs 的 Na(+)/H(+)交换器活性，并逆转了基因型之间的差异。cariporide 几乎完全消除了两种基因型的 Na(+)/H(+)交换器活性，并减弱了 LPS 诱导的 osr(WT) 小鼠细胞肿胀和 ROS 形成。总之，OSR1 部分缺乏会影响树突状细胞的 Na(+)/H(+)交换器活性、ROS 形成和迁移。
