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动态平衡 在靶组织中诱导转录和转录后修饰。甲状旁腺激素刺激心脏的吸收 骨骼中的矿物质基质，并增加活性维生素D、钙重吸收和磷酸盐的产生 肾脏的排泄物。尽管甲状旁腺激素作为调节荷尔蒙的核心作用，但许多下游 协调甲状旁腺素信号事件的细胞内靶分子尚未确定。特征描述 这些途径将扩大目前关于矿物质动态平衡机制的知识，并提示 新的治疗策略，以抵消或恢复甲状旁腺素在骨骼和肾脏疾病中的作用。最近，盐- 诱导蛋白激酶(SIKs)被认为是骨细胞甲状旁腺素信号的细胞内介质，而小分子蛋白激酶(SIKs)是骨细胞甲状旁腺素信号转导的调节因子。 SIKS的分子抑制剂在体内被发现模拟甲状旁腺激素对骨的作用。这项建议的目的是 探讨类似的依赖SIK的甲状旁腺素信号通路在肾脏中是否活跃。目标1 提案将探索PTH/SIK信号调节CYP27B1的分子机制 表情。在肾细胞、人肾器官和小鼠中，PTH和SIK抑制剂YKL-05-099 增加细胞色素P27B1的表达，这是一种将非活性维生素D转化为活性形式的酶。在肾脏细胞中， PTH处理导致SIK3底物CRTC2的磷酸化水平降低。剩余的中介人 PTH/Sik/CyP27B1途径将通过针对上游分子的体外实验来表征 在SIK下游的肾细胞和类器官中。CRTC家族成员的染色质免疫沉淀 将确定PTH/SIK转录激活所需的调节区。目标2将 明确PTH/SIK信号在肾脏磷酸盐重吸收中的作用。PTH或YKL-05-099处理的小鼠 降低血磷水平，YKL-05-099治疗降低磷酸盐转运体Npt2a 定位于肾刷状缘膜。将对这些小鼠进行测试，以确认SIK抑制会诱导 体内的磷酸尿症。三个SIK家族成员将在肾上皮细胞中被敲除及其对 磷酸盐吸收评估。目的3将描述缺乏肾脏SIK1和SIK3的小鼠的表型，以及 探讨小分子SIK抑制剂在CKD-MBD中的治疗潜力。这里描述的实验 探索SIKS在调节肾脏甲状旁腺激素信号转导中的新作用。此外，为 该项目将有助于阐明控制矿物质动态平衡的关键骨骼/肾脏调节轴。 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 在自身免疫和其他炎症性疾病中的应用，作为慢性疾病晚期的治疗 肾脏疾病(CKD)和其他对甲状旁腺激素无反应的矿物质离子稳态紊乱。