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

摘要 在肾脏中，Henle袢的粗升支（TAL）对NaCl稳态和血液循环至关重要。 压力调节在盐敏感性高血压的人类和动物模型中， 在TAL中增加，其中NaCl吸收取决于肾转运蛋白NKCC 2，顶端Na+/K+/2Cl- 协同转运体我们发现TAL顶膜上NKCC 2的存在控制了NaCl的吸收 在这个上皮中。控制顶端膜NKCC 2水平的分子机制涉及内吞作用， 再循环和胞外插入。内吞修复的抑制导致NKCC 2在膜上积聚 增加NaCl吸收。任何影响NKCC 2内吞作用的基因或蛋白质都可能影响 NKCC 2活性和肾盐转运，但已知只有少数蛋白质结合NKCC 2。使用靶向 通过蛋白质组学筛选，我们确定ALMS 1（Alström综合征1）和ACTN 4（α-辅肌动蛋白4）相互作用 NKCC 2的合作伙伴。我们还发现ALMS 1和ACTN 4相互作用，这增加了ALMS 1和ACTN 4相互作用的可能性。 这些蛋白质形成复合物。ALMS 1和ACTN 4的单核苷酸多态性与 高血压和肾功能下降。我们发现，ALMS 1基因敲除大鼠的表面NKCC 2 还有高血压我们发现，ACTN 4，一种参与足细胞生物学的蛋白质，也表达于 包括TAL。ALMS 1和ACTN 4在TAL处理肾脏NaCl中的作用 其在血压调节中的作用尚不清楚。我们假设ALMS 1控制表面NKCC 2 通过结合NKCC 2和ACTN 4的羧基末端来介导NKCC 2介导的NaCl吸收， NKCC 2从顶膜的内吞作用。TAL中ALMS 1或ACTN 4表达的减少 增加表面NKCC 2、NKCC 2介导的NaCl重吸收、肾小管-肾小球反馈（TGF）敏感性 导致盐敏感性高血压我们的长期目标是提高我们对 ALMS 1和ACTN 4在肾脏NaCl转运中的作用

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.