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

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.

摘要 DCT重构与人类疾病、饮食盐摄入量的改变和药物有关 行政管理。家族性高血钾高血压与利尿剂抵抗的关系 肥大和增生；而Gitelman综合征和East综合征导致DCT萎缩。然而，尽管它的 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 亚段，早期DCT1，晚期DCT2。Knepper和他的同事使用单细胞转录组学，最近 在DCT1中发现了一种罕见的增殖细胞群，他们认为这可能是导致 DCT独特的可塑性。我们实验室的初步数据显示，饮食中缺钾增加了 这些增殖细胞在DCT中所占比例大，并引起DCT肥大，尤其是沿DCT1。 此外，这项研究还表明，增殖期DCT细胞类型的转运蛋白转录水平较低 和更高水平的增殖转录本，表明DCT1细胞去分化为更具增殖性的 州政府。库林-环-连接酶(CRL)是一类E3泛素连接酶家族，介导调节细胞的降解。 蛋白质参与许多细胞功能，对细胞维持和清除多余的物质非常重要 蛋白质。最近发现CRL通过蛋白酶体降解with-NO-α来调节血压。 赖氨酸激酶(WNKS)。CUL3(Cullin 3)是CRLS的关键成分，它增加了泛素部分的靶向性 蛋白酶体降解的蛋白质。CUL3的突变导致了FHHt。所有CRL均由 COP9信号体(CSN)，与CRL相互作用，关闭泛素连接酶活性。疾病-- 导致CUL3突变抑制了CUL3与CSN的相互作用，从而离开了CUL3 过度活跃。为了研究受损的CSN-CUL3相互作用在人类疾病中的作用，我们灭活了 CSN通过沿整个肾单位删除Jab1(关键的CSN催化亚基)。尽管CUL3和CUL3 这些小鼠(KS-Jab1-/-)仅沿肾单位重塑 远端肾单位，DCT缩短，DCT1特异性蛋白大量减少。在这里，我们计划测试 我们的假设是CSN功能障碍导致DCT1细胞去分化，从而导致DCT重构 最新的技术进步和新的小鼠模型，使我们能够研究DCT在蛋白质和 史无前例的详细记录。我们利用DCT的荧光激活核分选(FANS)- 特定完整(在特定细胞类型中标记的核的分离)报告小鼠，具有可诱导的核 GFP的表达将大大丰富DCT细胞。我们将其与单核rna测序(snrna-seq)配对。 这就产生了关于DCT细胞群体的非常细粒度的数据。建议使用Jab1-/-进行的实验 小鼠将揭示DCT的可塑性，有助于揭示DCT重塑的机制 由CSN功能障碍引起。这将揭示有价值的信息，可能具有临床和生物学意义 在DCT重塑的情况下推进治疗的重要性。