Tissue-Specific Regulation and Effects of CYP24A1

项目摘要 正常的矿物质平衡是由甲状旁腺激素（PTH），成纤维细胞生长因子23 （FGF 23）和维生素D对肠、肾和骨的作用。维生素D受PTH和FGF 23：PTH的反调节 增加和FGF 23降低循环水平的生物活性形式的维生素D，1，25-二羟维生素 D（1，25 D），通过对1，25 D合成酶CYP 27 B1（1α-羟化酶）和1，25 D- 分解代谢酶，CYP 24 A1（24-羟化酶）。尽管有令人信服的人类遗传和生理证据 和动物，指出CYP 24 A1的至关重要性，大多数先前的研究维生素D在健康和 慢性肾脏病（CKD）主要关注CYP 27 B1合成1，25 D; CYP 24 A1对1，25 D的催化作用。为了推进CYP 24 A1的研究，我们开发了新的lox-P小鼠， Cyp 24 a1的组织选择性和诱导性缺失。我们将使用这种新颖的鼠标线来测试我们的总体 假设CYP 24 A1在肾脏和肾脏中的组织特异性作用有助于正常和紊乱 矿物质平衡由于肾脏是循环1，25 D的主要调节器，我们假设肾脏- 特异性Cyp 24 a1缺失将增加血清1，25 D和下游维生素D受体（VDR）活性， 组织，包括肠和肾。在CKD中，这将导致高钙血症，抑制PTH，但 进一步损害肾功能，由于1，25 D中毒。相反，由于肠道不参与 对于循环的1，25 D，我们预期丝氨酸特异性Cyp 24 a1缺失会增加1，25 D和VDR活性 只在肠道里由此导致的肠钙吸收增加将抑制PTH，这将导致 降低血清1，25 D，从而降低肾脏中的VDR活性。因此，我们假设， CKD患者的肠道CYP 24 A1将减轻继发性甲状旁腺功能亢进，而不引起高钙血症， 肾毒性在目的1中，我们将研究CYP 24 A1在正常矿物质稳态中的作用， 从肾脏（Six 2Cre-Cyp 24 flox）、肠（VillinCreERT 2-Cyp 24 flox）和整体缺失Cyp 24 a1的小鼠 （UBCCreERT2-Cyp24flox）。在目标2中，我们将检验我们的假设，即抑制肠道CYP 24 A1将减弱 继发性甲状旁腺功能亢进和CKD下游并发症，但不加重CKD。在目标1和2中， 效果的读数将包括矿物质稳态和肾功能的纵向生理测量， VDR靶基因在肾脏和肠中的表达;在目标2中，我们还将评估心脏结构， 功能，以及肾脏、骨和血管组织学。在目标3中，我们将定义CYP 24 A1的分子机制 在小鼠和人类肠道类器官中使用药理学和遗传学方法调节肠细胞。 效应读数将包括细胞信号传导测定和Cyp 24 a1和VDR靶基因的表达。通过定义 使用我们的新小鼠模型，我们将揭示CYP 24 A1的组织特异性作用和分子调控， CKD和其他矿物质稳态紊乱综合征的新治疗策略。

PROJECT SUMMARY Normal mineral homeostasis is regulated by the actions of parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and vitaminD on the intestine,kidney andbone. Vitamin D is counter regulatedby PTHand FGF23: PTH increases andFGF23 decreases circulatinglevels of thebiologically active formof vitamin D, 1,25-dihydroxyvitamin D (1,25D), via opposing actions on the 1,25D-synthetic enzyme, CYP27B1 (1α-hydroxylase), and the 1,25D- catabolic enzyme, CYP24A1 (24-hydroxylase). Despite compelling genetic and physiological evidence in humans and animals that point to the critical importance of CYP24A1, most prior research of vitamin D in health and in chronic kidney disease (CKD) focused on 1,25D synthesis by CYP27B1; much less attention has been devoted to 1,25D catabolism by CYP24A1. To advance research of CYP24A1, we developed new lox-P mice that enable tissue-selective and inducible deletion of Cyp24a1. We will use this novel mouse line to test our overarching hypothesis that tissue-specific effects of CYP24A1 in the kidney and intestinecontribute to normal and disordered mineral homeostasis. Since the kidney is the main regulator of circulating 1,25D, we hypothesize that kidney- specific Cyp24a1 deletion will increase serum 1,25D and downstream vitamin D receptor (VDR) activity in all tissues, including the intestine and kidney. In CKD, this will cause hypercalcemia that suppresses PTH, but will further harm kidney function due to 1,25D intoxication. In contrast, since the intestine does not contribute meaningfully to circulating1,25D, we expect intestine-specific Cyp24a1 deletion to increase1,25D and VDR activity only in the intestine. Resultant increases in intestinal calcium absorption will suppress PTH, which will lead to decreased serum 1,25D and thus, decreased VDR activity in the kidney. As a result, we hypothesize that inhibiting intestinal CYP24A1 in CKD will attenuate secondary hyperparathyroidismwithout causing hypercalcemia or further kidney toxicity. In Aim 1, we will investigate the effects of CYP24A1 in normal mineral homeostasis by studying mice with deletion of Cyp24a1 from the kidney (Six2Cre-Cyp24flox), intestine (VillinCreERT2-Cyp24flox) and globally (UBCCreERT2-Cyp24flox). In Aim 2, we will test our hypothesis that inhibiting intestinal CYP24A1 will attenuate secondary hyperparathyroidism and downstream complications of CKD without worsening CKD. In Aims 1 and 2, readouts of effect will include longitudinal physiological measures of mineral homeostasis and kidney function, and expression of VDR target genes in the kidney and intestine; in Aim 2 we will also assess cardiac structure and function, and kidney, bone and vascular histology. In Aim 3, we will define the molecular mechanisms of CYP24A1 regulationin enterocytes usingpharmacological and genetic approaches in mouseand humanintestinal organoids. Readouts of effect will include cell signaling assays and expressionof Cyp24a1 and VDR target genes. By defining the tissue-specific effects and molecular regulation of CYP24A1 using our new mouse models, we will uncover novel therapeutic strategies for CKD and other syndromes of disordered mineral homeostasis.