Role of Renal and Intestinal AC6 and NHE3 for Phosphate Homeostasis

精确调节身体的磷酸盐水平是一项关键任务。几乎所有慢性肾脏病患者 慢性肾脏病（CKD）患者表现出与心血管死亡率增加相关的高磷酸盐血症。肾 近曲小管中Pi的重吸收受肾脏调节，需要成纤维细胞生长因子23 （FGF 23）和甲状旁腺激素（PTH）。后者已被描述为通过环腺苷信号 腺苷酸环化酶（AC）产生的一磷酸腺苷（cAMP），并回收Na+-Pi共转运蛋白2a和2c （Npt 2a和Npt 2c）和Na+/H+交换器3（NHE 3）。的首要目标 本研究拟通过分析腺苷酸环化酶6（AC 6）和NHE 3在Pi稳态中的作用， 脑-肾轴我们发现AC 6是PTH介导的cAMP最重要的亚型 形成和Pi稳态。与预期的导致Pi排泄受损的病理生理学相反， AC 6的缺乏导致肾Pi消耗，其中80%的Npt 2a驻留在溶酶体中。为了避免进一步的Pi损失，PTH 预期FGF 23水平会受到抑制;然而，AC 6的缺乏与FGF 23水平显著相关。 这两种激素水平的升高表明这种Pi损失不能通过生殖途径来抵消。虽然监管 的内环境将需要肠道Pi摄取增强或不变，我们发现，缺乏 AC 6导致肠道Npt 2b几乎完全缺失。这种矛盾突出表明，AC 6在一个 迄今为止尚未确定的负反馈回路，抑制Pi调节激素。由于PTH也针对 NHE 3，我们产生了一种新的肾脏特异性NHE 3敲除小鼠，以确定NHE 3对NHE 3基因表达的贡献。 π稳态。虽然该模型具有正常的Npt 2a丰度，但Npt 2c丰度减少，提供了一个新的模型。 NHE 3和Npt 2c之间的新联系以前从未被证明过。在目标1中，我们将确定 在正常情况下和CKD下Pi稳态中的肾AC 6。在目标2中，我们将描述3 Pi稳态的特异性信号通路：i）Gαs蛋白偶联的AC 6/cAMP/蛋白激酶A; ii）Gαq/11 蛋白偶联磷脂酶C（PLC）/三磷酸肌醇/Ca 2 +/蛋白激酶C;和iii）FGF 23。一种新型 具有缺陷PLC的小鼠模型（命名为DSEL小鼠）和AC 6信号传导将使我们能够研究 这些途径中的每一种在调节近端小管中Npt 2a/c表达中的贡献。以确定 FGF 23信号传导的贡献，我们将通过一种新的中和作用来拮抗FGF 23， 抗体的目的3将确定NHE 3和Npt 2c之间是否存在用于Pi稳态的联系。基于 假设NHE 3受PTH调节，我们将使用我们的肾特异性NHE 3敲除小鼠来研究 Npt 2a/c运输和共定位，并确定在何种程度上NHE 3是需要肾Pi稳态。 目的4将通过利用新的方法来确定AC 6和/或NHE 3是否在肠Pi摄取和Pi稳态中起作用。 肠粘膜特异性AC 6和肠粘膜特异性NHE 3敲除小鼠。调节PTH、FGF 23 活性维生素D水平将测试Npt 2b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.