The role of salt inducible kinases in renal PTH action

盐诱导激酶在肾 PTH 作用中的作用

• 批准号: 10207599
• 负责人: Michael Mannstadt
• 金额: $ 41.09万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207599
• 关键词: 3-Dimensional; Acute; Adaptor Signaling Protein; Adenine; Autoimmune; Binding; Biology; Blood; Bone Diseases; Bone Resorption; Bypass; CYP27B1 gene; Calcium; Cells; Chronic; Chronic Kidney Failure; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Diet; Disease; Endocrine; Enzymes; Epithelial Cells; Event; Excretory function; Family; Family member; Future; Gene Expression; Genes; Genetic Transcription; Goals; Homeostasis; Hormonal; Hormone Responsive; Hormone use; Hormones; Human; In Vitro; Inflammatory; Injury to Kidney; Inorganic Phosphate Transporter; Ions; Kidney; Kidney Diseases; Knock-out; Knowledge; Light; Link; Mediating; Mediator of activation protein; Metabolism; Minerals; Mission; Modeling; Modification; Molecular; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nephrons; Nucleic Acid Regulatory Sequences; Oral; Organ; Organoids; Osteocytes; Osteogenesis; Osteoporosis; PTH gene; Parathyroid Hormone Receptor; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Inhibition; Play; Post-Translational Regulation; Production; Protein Isoforms; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Proximal Kidney Tubules; Receptor Activation; Regulation; Regulatory Pathway; Research; Role; Serum; Signal Pathway; Signal Transduction; Testing; Therapeutic; Therapeutic Uses; Tissues; Transcriptional Activation; United States National Institutes of Health; Upstream Enhancer; Vitamin D; bone; bone health; bone mass; brush border membrane; calcium metabolism; chromatin immunoprecipitation; experimental study; hormonal signals; hormone resistance; human disease; in vivo; inhibitor/antagonist; inorganic phosphate; kidney cell; kinase inhibitor; mouse model; novel; novel strategies; novel therapeutic intervention; receptor; renal epithelium; salt-inducible kinase; skeletal; skeletal disorder; small molecule; small molecule inhibitor; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; treatment effect; uptake; urinary

Project Summary Parathyroid hormone (PTH) maintains mineral ion homeostasis by interacting with the PTH/PTHrP receptor to induce transcriptional and post-transcriptional modifications in target tissues. PTH stimulates resorption of the mineral matrix in bone, and increases production of active vitamin D, calcium reabsorption, and phosphate excretion in the kidneys. Despite the central role of PTH as a regulatory hormone, many of the downstream intracellular target molecules that orchestrate PTH signaling events have yet to be identified. Characterization of these pathways will expand current knowledge of the mechanisms governing mineral homeostasis, and suggest new therapeutic strategies to counteract or restore PTH action in skeletal and renal disease. Recently, salt- inducible kinases (SIKs) were identified as intracellular mediators of PTH signaling in osteocytes, and small- molecule inhibitors of SIKs were found to mimic PTH action on bone in vivo. The objective of this proposal is to explore whether analogous SIK-dependent PTH signaling pathways are active in the kidney. Aim 1 of this proposal will explore the molecular mechanisms through which PTH/SIK signaling regulates CYP27B1 expression. In renal cells, human kidney organoids, and mice, both PTH and the SIK inhibitor YKL-05-099 increase expression of CYP27B1, the enzyme that converts inactive vitamin D to its active form. In renal cells, PTH treatment results in decreased phosphorylation of SIK3 substrate CRTC2. The remaining intermediaries of the PTH/SIK/CYP27B1 pathway will be characterized using in vitro experiments targeting molecules upstream and downstream of SIK in renal cells and organoids. Chromatin Immunoprecipitation of CRTC family members will identify the regulatory regions necessary for CYP27B1 transcriptional activation by PTH/SIK. Aim 2 will define the role of PTH/SIK signaling in renal phosphate reabsorption. Mice treated with PTH or YKL-05-099 have decreased serum phosphate levels, and YKL-05-099 treatment decreases phosphate transporter Npt2a localization to the renal brush border membrane. These mice will be tested to confirm that SIK inhibition induces phosphaturia in vivo. Three SIK family members will be knocked out in renal epithelial cells and their effect on phosphate uptake assessed. Aim 3 will characterize the phenotype of mice lacking renal SIK1 and SIK3, and explore the therapeutic potential of small molecule SIK inhibitors in CKD-MBD. The experiments described here explore a novel role for SIKs in regulating PTH signaling in the kidney. In addition, mouse models developed for this project will help to shed light on the crucial bone/kidney regulatory axis that controls mineral homeostasis and bone health. This project also offers a novel application for SIK inhibitors, a class of drug currently studied for their application in autoimmune and other inflammatory disorders, as treatment in later stages of chronic kidney disease (CKD) and other disorders of mineral ion homeostasis that are unresponsive to PTH.

项目摘要 甲状旁腺激素（PTH）通过与PTH/PTHrP受体相互作用维持矿物质离子稳态， 在靶组织中诱导转录和转录后修饰。甲状旁腺素刺激骨吸收 骨矿物质基质，并增加生产活性维生素D，钙重吸收，和磷酸盐 肾脏中的排泄物。尽管PTH作为一种调节激素发挥着重要作用，但许多下游调节激素的作用仍然存在。 协调PTH信号传导事件的细胞内靶向分子还有待鉴定。表征 这些途径将扩展目前对矿物质稳态机制的认识，并表明 在骨骼和肾脏疾病中抵消或恢复PTH作用的新治疗策略。最近，盐- 诱导激酶（SIKs）被鉴定为骨细胞中PTH信号传导的细胞内介质，而小- 发现SIKs的分子抑制剂在体内模拟PTH对骨的作用。这项建议的目的是 探索类似的依赖于SIK的PTH信号通路是否在肾脏中活跃。目标1 该提案将探讨PTH/SIK信号调节CYP 27 B1的分子机制 表情在肾细胞、人肾类器官和小鼠中，PTH和SIK抑制剂YKL-05-099 增加CYP 27 B1的表达，该酶将非活性维生素D转化为活性形式。在肾细胞中， PTH处理导致SIK 3底物CRTC 2的磷酸化降低。其余的中间人 将使用针对上游分子的体外实验来表征PTH/SIK/CYP 27 B1途径 以及肾脏细胞和类器官中的SIG下游。CRTC家族成员的染色质免疫沉淀 将确定PTH/SIK激活CYP 27 B1转录所需的调控区域。目标2将 确定PTH/SIK信号在肾磷酸盐重吸收中的作用。用PTH或YKL-05-099治疗的小鼠具有以下特征： 血清磷酸盐水平降低，YKL-05-099治疗降低磷酸盐转运蛋白Npt 2a 定位于肾刷状缘膜。将对这些小鼠进行测试，以确认抑制SIK诱导 体内磷酸尿。三个SIK家族成员将在肾上皮细胞中被敲除，并且它们对 磷酸盐吸收评估。目的3将表征缺乏肾SIK 1和SIK 3的小鼠的表型， 探索小分子SIK抑制剂在CKD-MBD中的治疗潜力。这里描述的实验 探索SIKs在调节肾脏PTH信号传导中的新作用。此外，开发用于 该项目将有助于阐明控制矿物质体内平衡的关键骨/肾调节轴 和骨骼健康。该项目还为目前正在研究的一类药物-SIK抑制剂提供了一种新的应用 用于自身免疫性和其他炎症性疾病，作为慢性炎症晚期的治疗， 肾脏疾病（CKD）和其它对PTH无反应的矿物离子体内平衡紊乱。