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.

项目摘要 正常的矿物质稳态受甲状旁腺骑马（PTH），成纤维细胞生长因子的作用23 （FGF23）和肠道，肾脏和骨上的维生素。维生素D是反调节的PTHAND FGF23：PTH 增加和FGF23降低了生物学活性维生素D的循环水平，1,25-二羟基维生素 D（1,25d），通过对1,25D合成酶，CYP27B1（1α-羟化酶）和1,25d-的相反作用。 分解代谢酶，CYP24A1（24-羟化酶）。尽管人类中令人信服的遗传和身体证据 以及指出CYP24A1至关重要的动物，维生素D在健康和研究中的大多数研究 慢性肾脏疾病（CKD）着重于CYP27B1的1,25D合成；关注的注意力要少得多 CYP24A1的1,25D分解代谢。为了推进CYP24A1的研究，我们开发了新的Lox-P小鼠 CYP24A1的组织选择性和可诱导的缺失。我们将使用这种新颖的鼠标线来测试我们的总体 假设CYP24A1在肾脏中的组织特异性作用和无效的无效分支 矿物稳态。由于肾脏是循环1,25D的主要调节剂，因此我们假设肾脏 - 特定的CYP24A1缺失将增加血清1,25D和下游维生素D受体（VDR）活性 组织，包括肠和肾脏。在CKD中，这将导致抑制PTH的高钙血症，但会导致 由于1,25D的触觉，进一步损害了肾功能。相反，由于肠子没有贡献 有意义地循环1,25D，我们预计肠道特异性的CYP24A1删除将增加1,25D和VDR活动 仅在肠道中。由此导致肠钙吸收的增加将抑制PTH，这将导致 血清1,25D降低，因此肾脏的VDR活性降低。结果，我们假设抑制 CKD中的肠CYP24A1将减弱次级性甲状旁腺功能亢进，而不会引起高钙血症或进一步 肾脏毒性。在AIM 1中，我们将通过研究CYP24A1在正常的豆骨稳态中的影响 从肾脏（Six2cre-Cyp24flox），肠道（Villincreert2-Cyp24flox）和全球删除CYP24A1的小鼠 （UBCCREERT2-CYP24FLOX）。在AIM 2中，我们将检验我们的假设，即抑制肠道CYP24A1会减弱 CKD的继发性甲状旁腺功能亢进症和下游并发症而不担心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物理测量，以及 VDR靶基因在肾脏和肠中的表达；在AIM 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AIM 3中，我们将定义CYP24A1的分子机制 使用小鼠和人脑癌的药物和遗传方法调节肠上皮细胞。 效应的读数将包括细胞信号测定和CYP24A1和VDR靶基因的表达。通过定义 使用新小鼠模型的CYP24A1的组织特异性效应和分子调节，我们将发现 CKD和其他无序刺稳态综合症的新型热策略。