Tissue-Specific Regulation and Effects of CYP24A1

CYP24A1 的组织特异性调控和作用

• 批准号: 10580931
• 负责人: MYLES S WOLF
• 金额: $ 54.75万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580931
• 关键词: Address; Adenine; Animals; Attention; Attenuated; Biological Assay; Blood Vessels; Bone Diseases; CRISPR/Cas technology; CYP27B1 gene; Calcium; Cardiac; Cardiovascular Diseases; Catabolism; Cessation of life; Chronic Kidney Failure; Complication; Data; Diet; Dihydroxycholecalciferols; Disease; Ensure; Enterocytes; Enzymes; Fibroblast Growth Factor Receptors; Fracture; Genes; Genetic; Health; Heart; Histology; Homeostasis; Human; Human Genome; Hypercalcemia; Intestines; Intoxication; Investigation; Kidney; Kidney Diseases; Knockout Mice; Liver; Longitudinal Studies; Measures; Metabolic; Metabolic Bone Diseases; Minerals; Mixed Function Oxygenases; Molecular; Mus; Nephrolithiasis; Organ; Organoids; PTH gene; Phenotype; Physiological; Protein Isoforms; Regulation; Renal function; Research; Role; Secondary Hyperparathyroidism; Serum; Signal Pathway; Signal Transduction; Skeletal Development; Structure; Surgical Models; Syndrome; Tamoxifen; Testing; Tissues; Toxic effect; Variant; Vitamin D; Vitamin D3 Receptor; Wild Type Mouse; bone; calcium absorption; design; fibroblast growth factor 23; genetic approach; genome wide association study; in vivo; indexing; inhibitor; inorganic phosphate; loss of function mutation; mouse model; nephrotoxicity; novel; novel therapeutic intervention; novel therapeutics; synthetic enzyme

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.

项目总结 正常的矿物质动态平衡受甲状旁腺激素、成纤维细胞生长因子23的调节 (FGF23)，以及肠道、肾脏和骨骼中的维生素D。维生素D受PTH和FGF23的反向调节：PTH FGF23增加和降低循环中生物活性形式维生素D，1，25-二羟基维生素E的水平 D(1，25D)，通过与1，25D合成酶CyP27B1(1α-羟基酶)和1，25D-羟基酶的相反作用。 分解代谢酶，CYP24A1(24-羟基酶)。尽管有令人信服的人类遗传和生理证据 以及指出CYP24A1的关键重要性的动物，大多数先前关于维生素D对健康和人类健康的研究 慢性肾脏疾病(CKD)关注的是由CYP27B1合成的1，25D；对此关注较少 1，25D由CYP24A1分解代谢。为了推进CYP24A1的研究，我们开发了新的LOX-P小鼠，使其能够 CYP24A1的组织选择性和可诱导性缺失。我们将使用这条新颖的鼠标线来测试我们最重要的 细胞色素P24A1在肾脏和肠道的组织特异性作用与正常和紊乱有关的假说 矿物动态平衡。由于肾脏是循环1，25D的主要调节器，我们假设肾脏- 特异性CYP24A1缺失将增加ALL患者血清1，25D及其下游维生素D受体(VDR)活性 组织，包括肠道和肾脏。在慢性肾脏病中，这将导致高血钙症，从而抑制甲状旁腺素，但会 1，25D中毒进一步损害肾功能。相反，由于肠道不起作用 对于循环1，25D有意义的是，我们预计肠道特异的CYP24A1缺失会增加1，25D和VDR的活性 只在肠子里。由此导致的肠道钙吸收增加将抑制甲状旁腺素，这将导致 降低血清1，25D，从而降低肾脏VDR活性。因此，我们假设抑制 慢性肾脏病患者的肠道细胞色素P24A1可减轻继发性甲状旁腺功能亢进症，而不会引起高钙血症或进一步 肾毒性。在目标1中，我们将通过以下研究来研究CYP24A1在正常矿物质动态平衡中的作用 肾脏(Six2Cre-Cyp24Flox)、肠道(VillinCreerT2-Cyp24Flox)和整体CYP24A1缺失的小鼠 (UBCCreERT2-Cyp24flx)。在目标2中，我们将验证我们的假设，即抑制肠道细胞色素P24A1将减弱 继发性甲状旁腺功能亢进和CKD的下游并发症而不加重CKD。在目标1和目标2中， 效果读数将包括矿物质动态平衡和肾功能的纵向生理测量，以及 VDR靶基因在肾脏和肠道中的表达；在目标2中，我们还将评估心脏结构和 功能，肾脏，骨骼和血管组织学。在目标3中，我们将定义CYP24A1的分子机制 在小鼠和人类肠道器官中使用药理学和遗传学方法对肠细胞进行调节。 影响的读数将包括细胞信号分析以及CYP24A1和VDR目标基因的表达。通过定义 通过我们的新小鼠模型，我们将揭示CYP24A1的组织特异性效应和分子调控 CKD和其他矿物质动态平衡紊乱综合征的新治疗策略。