Regulation of NKCC2 and renal NaCl transport by protein-protein interactions

通过蛋白质-蛋白质相互作用调节 NKCC2 和肾脏 NaCl 转运

• 批准号: 10585141
• 负责人: Pablo A. Ortiz
• 金额: $ 60.22万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-20 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10585141
• 关键词: Acetazolamide; Affect; Alstrom syndrome; Animal Model; Apical; Binding; Biological; Biology; Biotinylation; Blood Pressure; Bumetanide; Carrier Proteins; Cells; Complex; Data; Distal; Duct (organ) structure; Endocytosis; Epithelium; Excretory function; Feedback; Focal and Segmental Glomerulosclerosis; Functional disorder; Gene Deletion; Genes; Genetic; Goals; Homeostasis; Human; Hypertension; Image; Ions; Kidney; Kidney Diseases; Knock-out; Limb structure; Macula densa; Mass Spectrum Analysis; Measures; Mediating; Membrane; Microscopy; Molecular; Monitor; Mutation; Natriuresis; Nephrons; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Physiology; Proteins; Proteomics; Rattus; Recycling; Regional Perfusion; Regulation; Renal function; Retrieval; Role; Single Nucleotide Polymorphism; Sodium Chloride; Structure of ascending limb of Henle' s loop; Surface; Thick; absorption; alpha Actinin; apical membrane; benzamil; blood pressure regulation; epithelial Na+ channel; human disease; human model; novel; patch clamp; podocyte; prevent; protein protein interaction; renal damage; salt balance; salt sensitive hypertension; symporter; thiazide

Abstract In the kidney, the thick ascending limb (TAL) of the loop of Henle is critical for NaCl homeostasis and blood pressure regulation. In humans and animal models of salt-sensitive hypertension, NaCl absorption is abnormally increased in the TAL, where NaCl absorption depends on the renal transporter NKCC2, an apical Na+/K+/2Cl- co-transporter. We showed that the presence of NKCC2 at the TAL apical membrane controls NaCl absorption in this epithelium. The molecular mechanisms that control apical membrane NKCC2 levels involve endocytosis, recycling and exocytic insertion. Inhibition of endocytic retrieval causes NKCC2 accumulation at the membrane and increased NaCl absorption. Any gene or protein affecting NKCC2 endocytosis could potentially influence NKCC2 activity and renal salt transport but only few proteins are known to bind NKCC2. Using a targeted proteomics screen, we identified ALMS1 (Alström Syndrome 1) and ACTN4 (alpha-actinin 4) as interacting partners of NKCC2. We also found that ALMS1 and ACTN4 interact with each other, raising the possibility that these proteins form a complex. Single nucleotide polymorphisms in ALMS1 and ACTN4 are associated with hypertension and decreased kidney function. We found that ALMS1 knockout rats have higher surface NKCC2 and high blood pressure. We found that ACTN4, a protein involved in podocyte biology, is also expressed throughout the nephron, including the TAL. The roles of ALMS1 and ACTN4 in renal NaCl handling by the TAL and their role in blood pressure regulation are unknown. We hypothesize that ALMS1 controls surface NKCC2 levels and NKCC2-mediated NaCl absorption by binding the carboxyl-terminus of NKCC2 and ACTN4 to mediate NKCC2 endocytosis from the apical membrane. A decrease in ALMS1 or ACTN4 expression in the TAL increases surface NKCC2, NKCC2-mediated NaCl reabsorption, tubulo-glomerular feedback (TGF) sensitivity and leads to salt-sensitive hypertension. Our long-term goal is to increase our understanding of the role of ALMS1 and ACTN4 in kidney NaCl transport.

抽象的 在肾脏中，亨利环的粗升肢 (TAL) 对于 NaCl 稳态和血液至关重要 在盐敏感性高血压的人类和动物模型中，氯化钠吸收异常。 TAL 增加，其中 NaCl 吸收取决于肾脏转运蛋白 NKCC2，即顶端 Na+/K+/2Cl- 我们发现 TAL 顶膜上 NKCC2 的存在控制着 NaCl 的吸收。 控制顶膜 NKCC2 水平的分子机制涉及内吞作用， 内吞回收的抑制导致 NKCC2 在膜上积累。 任何影响 NKCC2 内吞作用的基因或蛋白质都可能产生影响。 NKCC2 活性和肾盐转运，但已知只有少数蛋白质能够结合 NKCC2。 通过蛋白质组学筛选，我们确定 ALMS1（阿尔斯特罗姆综合征 1）和 ACTN4（α-辅肌动蛋白 4）具有相互作用 我们还发现 ALMS1 和 ACTN4 相互作用，这增加了以下可能性： 这些蛋白质形成复合物，与 ALMS1 和 ACTN4 中的单核苷酸多态性相关。 我们发现 ALMS1 敲除大鼠的表面 NKCC2 较高。 我们发现，ACTN4（一种参与足细胞生物学的蛋白质）也有表达。 整个肾单位，包括 TAL ALMS1 和 ACTN4 在 TAL 处理肾 NaCl 中的作用。 它们在血压调节中的作用尚不清楚。我们研究发现 ALMS1 控制表面 NKCC2。 通过结合 NKCC2 和 ACTN4 的羧基末端介导的水平和 NKCC2 介导的 NaCl 吸收 顶膜 NKCC2 内吞作用 TAL 中 ALMS1 或 ACTN4 表达减少。 增加表面 NKCC2、NKCC2 介导的 NaCl 重吸收、肾小管肾小球反馈 (TGF) 敏感性 并导致盐敏感性高血压。我们的长期目标是增加我们对盐敏感性高血压的作用的了解。 ALMS1 和 ACTN4 在肾脏 NaCl 转运中的作用。