SGK Regulation of Epithelial Sodium Transport

项目总结/摘要 肾小管中Na+和K+的调节性转运是血压调节和体液和 电解质稳态mTORC 2-SGK 1-ENaC轴是这种调节的成熟组分。 然而，关键的机械特征仍然表征不佳，并且体内数据有限。我们有 使用体内和体外方法来鉴定该信号系统的新特征，特别是其在K+ 体内平衡我们最近的研究表明：1）K+通过WNK激酶激活mTORC 2; 2）肾小管细胞 mTORC 2是小鼠对K+负荷的快速反应的核心; 3）mTORC 2的结构特征，阐明 使用cryo-EM，在mTORC 2活性和特异性中起重要作用。为了探索这些假设，我们将： 目的1：评估肾小管特异性敲除小鼠对K+反应的时间顺序。 mTORC 2。我们已经产生了一个快速诱导的肾小管特异性Rictor KO（TRKO）小鼠模型。 初步数据显示，这些小鼠不能正常响应KCl; WT和KO小鼠之间的差异 在KCl管饲后<3 h表现出，并且在高钾饮食48 h时显著。为了描述这些小鼠，我们将 确定：（A）对急性KCl负荷的反应时间过程。小鼠将用口服K+负荷和尿K+负荷处理。 并评估血浆参数。将收获肾脏并评估组织信号传导 参数和离子转运蛋白的表达和修饰。ENaC、ROMK和BK渠道活动将在 使用膜片钳测量。将捕获来自补丁细胞的细胞质，并对mRNA进行RNA-seq分析。 来鉴定单细胞基因表达模式。(B)基因缺失的时间过程：小鼠将适应高水平的基因缺失。 K+饮食开始基因删除之前。将对肾上腺完整和ADX + aldo进行平衡实验 在表型出现之前和出现期间的时间点对小鼠进行免疫。将进行膜片钳检查， 如（A）中那样收获和分析RNA。(C)评估PKC的作用并确定新的靶点。我们将确定关键 PKC底物，并在体内和培养细胞中对其进行功能表征。目标2：mTORC 2 分子信号机制和特异性：WNK激酶的结构特征和作用。我们将 使用培养的细胞建立mTORC 2调节SGK 1的细胞和分子特征， 冷冻电镜(A)研究WNK 1和WNK 4作为促进mTORC 2依赖性 SGK 1以K+依赖的方式磷酸化。我们将研究WNK激酶对身体的影响， SGK 1的相互作用和磷酸化，以及对培养的mpkCCD中ENaC和ROMK的功能影响， HEK-293细胞。(B)mTORC 2核心复合物的Cryo-EM结构分析，以及SGK 1和WNK 1。 mTORC 2核心组件将在存在和不存在SGK 1和WNK的情况下接受冷冻EM 激酶。目的3：评估WNK 1和WNK 4的作用以及与mTORC 2的体内功能相互作用 使用诱导型KO模型。WNK 1和WNK 4诱导型KO小鼠的特征与TRKO相似， 目的1A;将研究Na+和K+的处理以及生化特征。

PROJECT SUMMARY/ABSTRACT Regulated transport of Na+ and K+ in the kidney tubules is central to blood pressure regulation and fluid and electrolyte homeostasis. The mTORC2-SGK1-ENaC axis is a well-established component of this regulatory machinery, however, key mechanistic features remain poorly characterized, and in vivo data is limited. We have used in vivo and in vitro approaches to identify novel features of this signaling system, particularly its role in K+ homeostasis. Our recent data suggest that: 1) K+ acts through WNK kinases to activate mTORC2; 2) tubule cell mTORC2 is central to the rapid response to a K+ load in mice; 3) Structural features of mTORC2, elucidated using cryo-EM, play an essential role in mTORC2 activity and specificity. To explore these hypotheses, we will: Aim 1: Assess the temporal sequence of responses to K+ in mice with tubule-specific knockout of mTORC2. We have generated a rapidly inducible kidney tubule-specific Rictor KO (TRKO) mouse model. Preliminary data show that these mice fail to respond to KCl normally; differences between WT and KO mice manifest in <3h following KCl gavage, and are striking by 48 h of high K diet. To characterize these mice, we will determine: (A) Time course of response to acute KCl load. Mice will be treated with an oral K+ load and urinary and plasma parameters will be assessed. Kidneys will be harvested and assessed for tissue signaling parameters and ion transporter expression and modification. ENaC, ROMK, and BK channel activities will be measured using patch clamp. Cytoplasm from patched cells will be captured and mRNA subjected to RNA-seq to identify single-cell gene expression patterns. (B) Time course of gene deletion: Mice will be adapted to a high K+ diet prior to initiating gene deletion. Balance experiments will be performed on adrenal-intact and ADX + aldo mice at time points prior to and during emergence of phenotype. Patch clamp will be performed and cytoplasmic RNA harvested and analyzed as in (A). (C) Evaluate PKC role and identify novel targets. We will identify key PKC substrates using LC/MS, and functionally characterize them in vivo and in cultured cells. Aim 2: mTORC2 molecular signaling mechanisms and specificity: structural features and effects of WNK kinases. We will establish the cellular and molecular features that underlie mTORC2 regulation of SGK1 using cultured cells and cryo-EM. (A) Investigate WNK1 and WNK4 as scaffolds that promote mTORC2-dependent phosphorylation of SGK1 in a K+-dependent fashion. We will examine effects of WNK kinases on physical interactions and phosphorylation of SGK1, and functional effects on ENaC and ROMK in cultured mpkCCD and HEK-293 cells. (B) Cryo-EM structural analysis of mTORC2 core complex, and with SGK1 and WNK1. mTORC2 core components will be subjected to cryo-EM in the presence and absence of SGK1 and WNK kinases. Aim 3: Assess the roles of WNK1 and WNK4 and functional interactions with mTORC2 in vivo using inducible KO models. WNK1 and WNK4 inducible KO mice will be characterized similarly to TRKO in aim 1A; Na+ and K+ handling, and biochemical features will be investigated.