The role of the COP9 signalosome in distal nephron remodeling

COP9信号体在远端肾单位重塑中的作用

• 批准号: 10723624
• 负责人: Ryan J Cornelius
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723624
• 关键词: Atrophic; Biological; Blood Vessels; COPS5 gene; Carrier Proteins; Catalytic Domain; Cell Maintenance; Cell Nucleus; Cell physiology; Cells; Clinical; Cullin Proteins; Data; Dietary Potassium; Disease; Distal; Distal convoluted renal tubule structure; Diuretics; Familial disease; Family; Fluorescence; Functional disorder; Genes; Genetic Transcription; Hyperplasia; Hypertrophy; Impairment; Kidney; Knock-out; Lead; Ligase; Light; Lysine; Mediating; Modeling; Molecular; Mus; Mutation; Nephrons; Nuclear; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Physiological; Population; Potassium; Potassium Deficiency; Proteins; Reporter; Research; Resistance; Resolution; Role; SLC11A2 gene; SLC12A3 gene; Sodium; Sorting; Specificity; Stimulus; Syndrome; Techniques; Technology; Testing; Transcript; Ubiquitin; Work; blood pressure regulation; career; career development; cell dedifferentiation; cell type; clinically significant; cullin-3; design; dietary salt; experimental study; familial hyperkalemic hypertension; human disease; kidney cortex; mouse model; novel; protein degradation; response; salt intake; single nucleus RNA-sequencing; transcriptome; transcriptome sequencing; transcriptomics; ubiquitin ligase; ubiquitin-protein ligase; Atrophic; Biological; Blood Vessels; COPS5 gene; Carrier Proteins; Catalytic Domain; Cell Maintenance; Cell Nucleus; Cell physiology; Cells; Clinical; Cullin Proteins; Data; Dietary Potassium; Disease; Distal; Distal convoluted renal tubule structure; Diuretics; Familial disease; Family; Fluorescence; Functional disorder; Genes; Genetic Transcription; Hyperplasia; Hypertrophy; Impairment; Kidney; Knock-out; Lead; Ligase; Light; Lysine; Mediating; Modeling; Molecular; Mus; Mutation; Nephrons; Nuclear; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Physiological; Population; Potassium; Potassium Deficiency; Proteins; Reporter; Research; Resistance; Resolution; Role; SLC11A2 gene; SLC12A3 gene; Sodium; Sorting; Specificity; Stimulus; Syndrome; Techniques; Technology; Testing; Transcript; Ubiquitin; Work; blood pressure regulation; career; career development; cell dedifferentiation; cell type; clinically significant; cullin-3; design; dietary salt; experimental study; familial hyperkalemic hypertension; human disease; kidney cortex; mouse model; novel; protein degradation; response; salt intake; single nucleus RNA-sequencing; transcriptome; transcriptome sequencing; transcriptomics; ubiquitin ligase; ubiquitin-protein ligase

Abstract DCT remodeling has been associated with human diseases, changes in dietary salt intake, and drug administration. The disease familial hyperkalemic hypertension (FHHt) and diuretic resistance cause hypertrophy and hyperplasia; whereas, Gitelman and EAST syndromes lead to DCT atrophy. Yet despite its clinical significance, the cellular and molecular basis for this plasticity is unclear. The DCT comprises two subsegments, the early DCT1 the late DCT2. Knepper and colleagues, using single cell transcriptomics, recently identified a rare proliferative cell population within the DCT1, which they suggested may be responsible for the DCT's unique plasticity. Preliminary data from our lab showed that dietary potassium deficiency increased the percent of these proliferative cells in the DCT and caused DCT hypertrophy, especially along the DCT1. Furthermore, this work showed the proliferative DCT cell type to have lower levels of transport protein transcripts and higher levels of proliferative transcripts, suggesting that DCT1 cells dedifferentiate into a more proliferative state. Cullin-RING-ligases (CRLs) are a family of E3 ubiquitin ligases that mediate regulated degradation of proteins and are involved in many cellular functions important for cell maintenance and elimination of unwanted proteins. CRLs were recently discovered to regulate blood pressure via proteasomal degradation of with-no- lysine kinases (WNKs). Cullin 3 (CUL3) is the critical component of CRLs, which add ubiquitin moieties targeting proteins for proteasomal degradation. Mutations in CUL3 cause the disease FHHt. All CRLs are regulated by the COP9 signalosome (CSN), which interacts with the CRL and turns off ubiquitin ligase activity. Disease- causing CUL3 mutations inhibit the ability of CUL3 to interact with the CSN and therefore leave CUL3 hyperactivated. To investigate the role of impaired CSN-CUL3 interaction in human disease, we inactivated the CSN by deleting Jab1 (the key CSN catalytic subunit) along the entire nephron. Despite the fact that both CUL3 and the CSN are expressed all along the nephron, these mice (KS-Jab1-/-) showed remodeling only along the distal nephron, with shortening of the DCT and a large reduction in DCT1-specific proteins. Here, we plan to test our hypothesis that CSN dysfunction causes dedifferentiation of DCT1 cells leading to DCT remodeling using recent technological advances and new mouse models that allow us to study DCT remodeling at the protein and transcript level in unprecedented detail. We have utilized fluorescence-activated nucleus sorting (FANS) of DCT- specific INTACT (Isolation of Nuclei Tagged in specific Cell Types) reporter mice, which have inducible nuclear GFP expression to greatly enrich for DCT cells. We pair this with single nucleus RNA sequencing (snRNA-seq) that generates remarkably granular data about DCT cell populations. The proposed experiments with Jab1-/- mice will shine a light on the plasticity of the DCT, helping to uncover the mechanisms for DCT remodeling caused by CSN dysfunction. This will reveal valuable information that could have clinical and biological importance in advancing therapies in cases where the DCT remodels.

抽象的 DCT重塑与人类疾病，饮食盐摄入的变化和药物有关 行政。疾病家族性高钾血高血压（FHHT）和利尿剂抗性导致 肥大和增生；而Gitelman和East综合征导致DCT萎缩。尽管有它 临床意义，这种可塑性的细胞和分子基础尚不清楚。 DCT包括两个 子策略，DCT1早期DCT2。最近使用单细胞转录组学的膝盖和同事 确定了DCT1中罕见的增殖细胞群，他们认为这可能是负责的 DCT的独特可塑性。我们实验室的初步数据表明，饮食中的钾缺乏症增加了 这些增殖细胞中的百分比在DCT中并引起DCT肥大，尤其是沿DCT1。 此外，这项工作显示了增殖的DCT细胞类型的转运蛋白转录本水平较低 以及更高水平的增殖转录本，表明DCT1细胞将其推导成更加增殖 状态。 cullin-ring-ligases（CRL）是E3泛素连接酶的家族，可介导调节的降解的降解 蛋白质，并参与许多细胞功能，对于细胞维持和消除不需要 蛋白质。最近发现CRL通过蛋白酶体降解来调节血压 赖氨酸激酶（WNK）。 Cullin 3（Cul3）是CRL的关键组成部分，它添加了泛素部分的靶向 蛋白质降解的蛋白质。 CUL3中的突变引起疾病FHHT。所有CRL均由 与CRL相互作用并关闭泛素连接酶活性的COP9信号体（CSN）。疾病- 引起CUL3突变抑制CUL3与CSN相互作用的能力，因此离开CUL3 过度活化。为了研究CSN-CUL3相互作用受损在人类疾病中的作用，我们将 CSN通过删除整个肾单位的JAB1（键CSN催化亚基）。尽管两个Cul3都 CSN沿着肾单位始终表达，这些小鼠（ks-jab1 - / - ）仅沿着 远端肾单位，DCT缩短，DCT1特异性蛋白质大幅降低。在这里，我们计划测试 我们假设CSN功能障碍会导致DCT1细胞的去分化，从而导致DCT重塑使用 最新的技术进步和新的小鼠模型，使我们能够研究蛋白质和 成绩单级别以前所未有的细节。我们已经使用了DCT-的荧光激活的核分选（FAN） 特定的完整（在特定细胞类型中标记的核的分离）记者小鼠，它们具有诱导核 GFP表达极大地富含DCT细胞。我们将其与单核RNA测序（SNRNA-Seq）配对 这会产生有关DCT细胞群体的颗粒状数据。提出的JAB1 - / - 的实验 小鼠会照亮DCT的可塑性，有助于发现DCT重塑的机制 由CSN功能障碍引起。这将揭示可能具有临床和生物学的宝贵信息 在DCT重塑的情况下，重要的是推进疗法。