Role of Renal and Intestinal AC6 and NHE3 for Phosphate Homeostasis

精确调节人体的磷酸盐水平是一项关键任务。几乎所有的慢性肾脏病患者 疾病(CKD)表现为高磷血症，这与心血管死亡率的增加有关。肾 PI在近曲小管的重吸收受激素调节，需要成纤维细胞生长因子23 (FGF23)和甲状旁腺激素(PTH)。后者已被描述为通过环腺苷传递信号 腺苷环化酶(AC)产生的单磷酸(CAMP)和回收Na+-PI共转运体2a和2c (Npt2a和Npt2c)和Na+/H+交换器3(NHE3)。的首要目标是 本研究旨在通过分析腺酰环化酶6(AC6)和NHE3在磷脂酰肌醇(PI)内稳态中的作用 肠-肾轴。我们发现AC6是甲状旁腺素介导的cAMP的最重要的亚型 形成和等电点动态平衡。与导致PI排泄受损的预期病理生理学相反， 缺乏AC6导致肾脏PI减少，80%的Npt2a驻留在溶酶体中。为了避免进一步的PI损失，PTH 预计FGF23水平将被抑制；然而，缺乏AC6与显著相关 这两种激素水平的升高表明，这种PI的丧失不能被激素所抵消。在监管期间 的环境需要增强或保持肠道PI摄取，我们发现缺乏 AC6导致肠道Npt2b几乎完全缺失。这一矛盾突出了AC6在一种 到目前为止，抑制PI调节激素的负反馈环尚不清楚。因为PTH也针对 NHE3，我们建立了一种新的肾脏特异性NHE3基因敲除小鼠，以确定NHE3对 PI动态平衡。虽然该模型具有正常的Npt2a丰度，但Npt2c丰度减少，提供了 NHE3和Npt2c之间的新联系以前从未显示过。在目标1中，我们将确定 肾脏AC6在正常和CKD状态下的PI动态平衡。在目标2中，我们将勾勒出3 PI稳态的特定信号通路：I)GαS蛋白偶联AC6/cAMP/蛋白激酶A；II)GαQ/11 蛋白偶联磷脂酶C(PLC)/三磷酸肌醇/钙/蛋白激酶C；以及iii)FGF23。一本小说 PLC缺陷的小鼠模型(命名为DSEL小鼠)和AC6信号将使我们能够研究 这些途径中的每一个在调节Npt2a/c在近端小管中的表达中的作用。要确定 FGF23信号转导途径的作用我们将通过一种新的中和机制在药理上拮抗FGF23 抗体。目标3将确定NHE3和Npt2c之间是否存在PI动态平衡的连锁。基于 假设NHE3受甲状旁腺素调节，我们将使用我们的肾脏特异性NHE3基因敲除小鼠来研究 Npt2a/c的转运和共定位，并确定NHE3在多大程度上需要肾脏PI的动态平衡。 目标4将确定AC6和/或NHE3是否在肠道PI摄取和PI动态平衡中发挥作用 肠粘膜特异性AC6和肠粘膜特异性NHE3基因敲除小鼠。调制PTH、FGF23 活性维生素D水平将测试Npt2b的调节。

The precise regulation of the body's phosphate level is a critical task. Nearly all patients with chronic kidney disease (CKD) exhibit hyperphosphatemia which is associated with increased cardiovascular mortality. Renal reabsorption of Pi in the proximal tubule is hormonally regulated and requires fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). The latter has been described to signal via cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclases (AC), and retrieve Na+-Pi cotransporters 2a and 2c (Npt2a and Npt2c) and Na+/H+ exchanger 3 (NHE3) from the apical cell membrane. The overarching goal of this proposal is to determine the roles of adenylyl cyclase 6 (AC6) and NHE3 in Pi homeostasis by analyzing the intestine-kidney axis. We identified that AC6 is the most important isoform for PTH-mediated cAMP formation and Pi homeostasis. In contrast to the expected pathophysiology resulting in impaired Pi excretion, lack of AC6 causes renal Pi wasting with 80% of Npt2a residing in lysosomes. To avoid further Pi loss, PTH and FGF23 levels would be expected to be suppressed; however, lack of AC6 is associated with significantly elevated levels of both hormones indicating that this Pi loss cannot be countered hormonally. While regulation of the milieu intérieur would require intestinal Pi uptake to be enhanced or unchanged, we found that lack of AC6 causes an almost complete absence of intestinal Npt2b. This paradox highlights that AC6 plays a role in a so far unidentified negative feedback loop that suppresses Pi regulating hormones. Since PTH also targets NHE3, we generated a novel kidney-specific NHE3 knockout mouse to determine the contribution of NHE3 for Pi homeostasis. While this model has normal Npt2a abundance, Npt2c abundance is diminished, providing a novel link between NHE3 and Npt2c that has never been shown before. In Aim 1, we will determine the role of renal AC6 in Pi homeostasis under normal conditions and CKD. In Aim 2, we will delineate the contribution of 3 specific signaling pathways for Pi homeostasis: i) Gαs protein coupled AC6/cAMP/protein kinase A; ii) Gαq/11 protein coupled phospholipase C(PLC)/inositol triphosphate/Ca2+/protein kinase C; and iii) FGF23. A novel mouse model with defective PLC (named DSEL mouse) and AC6 signaling will allow us to study the contribution of each of these pathways in regulating expression of Npt2a/c in the proximal tubule. To determine the contribution of FGF23 signaling we will pharmacologically antagonize FGF23 via a novel neutralizing antibody. Aim 3 will determine if there is a linkage between NHE3 and Npt2c for Pi homeostasis. Based on the hypothesis that NHE3 is regulated by PTH, we will use our kidney-specific NHE3 knockout mouse to study Npt2a/c trafficking and colocalization and determine to which extent NHE3 is required for renal Pi homeostasis. Aim 4 will determine if AC6 and/or NHE3 play a role in intestinal Pi uptake and Pi homeostasis by utilizing novel intestinal mucosa-specific AC6 and intestinal mucosa-specific NHE3 knockout mice. Modulating PTH, FGF23 and active vitamin D levels will test for the regulation of Npt2b.