Regulation of FGF23 in Chronic Kidney Disease (CKD) by iron and inflammation

 说明(申请人提供)：成纤维细胞生长因子23(FGF23)由骨细胞分泌，调节磷酸盐和维生素D的动态平衡。血清FGF23水平在慢性肾脏病(CKD)病程早期升高，高水平独立地与CKD进展、心血管疾病和死亡的更大风险相关。这些发现激发了人们对设计降低FGF23水平的治疗的兴趣，但这种方法受到对从早期CKD开始刺激FGF23产生的分子机制的缺乏了解的限制。缺铁是FGF23转录的一个新的刺激物，可能是CKD患者FGF23水平升高的关键因素。虽然早期慢性肾功能不全患者中明显的铁缺乏并不常见，但由于慢性炎症导致的网状内皮铁滞留所致的功能性铁缺乏非常普遍。在这项应用的初步数据中，我们证明了高水平的铁隔离蛋白，中性粒细胞明胶酶相关脂蛋白(NGAL)，降低了骨细胞内的铁浓度，并刺激了FGF23的产生。在进一步的初步数据中，我们报道了NGAL介导的细胞铁减少稳定了缺氧诱导因子(HIF)1a，HIF 1a通过结合FGF23启动子启动FGF23转录。由于肾脏损伤和炎症导致NGAL表达增加，CKD患者血清NGAL水平长期升高，我们认为肾脏损伤刺激NGAL的产生，从而诱导骨细胞铁外流，造成细胞内铁缺乏，从而稳定HIF1a并促进FGF23的产生。在这项创新的提案中，我们将研究炎症、NGAL以及真正的和功能性铁缺乏对健康和CKD患者FGF23产生的调节作用。在目标1中，我们将通过将野生型和NGALko小鼠暴露在三种全身炎症模型中来确定炎症和NGAL对FGF23产生的调节作用，并将证明内源性和外源性NGAL增加FGF23的产生。在目标2中，我们将研究HIF1a作为FGF23调节的分子介质，它是NGAL、铁、炎症和FGF23之间的机制串扰的基础。我们将进行芯片分析和定点突变来测试HIF1a是否与FGF23启动子结合，我们将在骨细胞中删除HIF1a，以证明HIF1aOc-CKO小鼠对NGAL和缺铁诱导的FGF23增加具有抵抗力。在目标3中，我们将通过研究NGAL和HIF1a缺失对进展性CKD模型Col4a3ko小鼠的影响，确定NGAL和HIF1a通路在CKD中的临床意义和治疗潜力。我们将研究Col4a3ko/NGALko和Col4a3ko/HIF1aOc-CKO复合突变体是否显示出相对于完整NGAL和HIF1a系统的Col4a3ko小鼠FGF23产生的钝性增加，我们还将测试NGAL阻断抗体和HIF1抑制剂对Col4a3ko小鼠FGF23产生的影响。该项目将为FGF23在健康和CKD中的分子调控提供新的见解，并支持我们开发新的治疗方法以改善CKD预后的最终目标。

 DESCRIPTION (provided by applicant): Fibroblast growth factor 23 (FGF23) is secreted by osteocytes and regulates phosphate and vitamin D homeostasis. Serum FGF23 levels rise early in the course of chronic kidney disease (CKD), and high levels are independently associated with greater risks of CKD progression, cardiovascular disease and death. These findings have stimulated interest in designing therapies to lower FGF23 levels, but the approach is limited by poor understanding of the molecular mechanisms that stimulate FGF23 production beginning in early CKD. Iron deficiency is a novel stimulator of FGF23 transcription and may be a critical contributor to elevated FGF23 levels in CKD. Although overt iron deficiency is uncommon in early CKD, functional iron deficiency due to reticuloendothelial iron sequestration as a result of chronic inflammation is highly prevalent. In preliminary data presented in this application, we demonstrate that high levels of the iron sequestering protein, neutrophil gelatinase associated lipocalin (NGAL), reduce intracellular iron concentrations in osteocytes and stimulate FGF23 production. In further preliminary data, we report that NGAL-mediated reductions in cellular iron stabilize hypoxia inducible factor (HIF)1a, which initiates FGF23 transcription by binding the FGF23 promoter. Since NGAL expression is increased in response to kidney injury and inflammation, and serum levels are chronically elevated in CKD, we propose that kidney injury stimulates NGAL production, which induces iron efflux from osteocytes, creating intracellular iron deficiency that stabilizes HIF1a and promotes FGF23 production. In this innovative proposal, we will examine the regulatory effects of inflammation, NGAL, and true and functional iron deficiency on FGF23 production in health and in CKD. In Aim 1, we will define the regulatory effects of inflammation and NGAL on FGF23 production by exposing wild type and NGALko mice to three models of systemic inflammation, and we will show that endogenous and exogenous NGAL increases FGF23 production. In Aim 2, we will investigate HIF1a as a molecular mediator of FGF23 regulation that underlies the mechanistic crosstalk between NGAL, iron, inflammation and FGF23. We will perform CHIP assays and site-directed mutagenesis to test whether HIF1a binds the FGF23 promoter, and we will delete HIF1a in osteocytes to demonstrate that HIF1aOc-cko mice are resistant to NGAL and iron deficiency-induced increases in FGF23. In Aim 3, we will determine the clinical relevance and therapeutic potential of the NGAL and HIF1a pathways in CKD by studying the impact of NGAL and HIF1a deletion in the Col4a3ko mouse model of progressive CKD. We will investigate if Col4a3ko/NGALko and Col4a3ko/HIF1aOc-cko compound mutants display blunted increases in FGF23 production relative to Col4a3ko mice with intact NGAL and HIF1a systems, and we will also test the effects on FGF23 production of NGAL blocking antibodies and HIF1 inhibitors in Col4a3ko mice. The project will contribute new insights into the molecular regulation of FGF23 in health and in CKD, and support our ultimate goal of developing novel therapeutic approaches to improve outcomes in CKD.