Polycystin Dependent Mechanisms of Tubular Plasticity

管状可塑性的多囊蛋白依赖性机制

基本信息

批准号：
10643823
负责人：
STEFAN SOMLO
金额：
$ 47.36万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10643823
关键词：
Address Adult Apoptotic Autosomal Dominant Polycystic Kidney Bacterial Artificial Chromosomes Biological Models Cell Culture Techniques Cell Lineage Cell Shape Cell model Cells Cilia Columnar Epithelium Complex Consensus Cre lox recombination system Cyst Cystic kidney Data Dialysis procedure Disease Enterobacteria phage P1 Cre recombinase Environment Epithelial Cells Fibrosis Gene Expression Profile Gene Silencing Genes Genetic Diseases Genetic Models Genetic Transcription Hepatic Cyst Human Inflammation Inherited Intuition Kidney Kidney Diseases Laboratories Modality Modeling Molecular Monitor Morphology Mus Mutation Nephrons Orthologous Gene Outcome PKD2 protein Pathogenesis Pathway interactions Patients Phenotype Physiological Polycystic Kidney Diseases Population Process Proliferating Recovery Renal function Reproducibility Research Resolution Role Stochastic Processes System Testing Therapeutic Time Tissues Transplantation Tubular formation Validation Work base cell type field study human disease in vivo inducible Cre innovation kidney preservation kidney repair loss of function man molecular modeling mouse model mutant novel polycystic liver disease pre-clinical programs repaired response single-cell RNA sequencing

项目摘要

Autosomal dominant polycystic kidney disease (ADPKD) results primarily from mutations in PKD1 and PKD2 encoding polcysytin-1 (PC1) and polycystin-2 (PC2), respectively. Since PKD1 and PKD2 were discovered, there has been significant progress in understanding the functions of the polycystins (PCs). Much recent progress has been based on in vivo orthologous gene mouse models which in turn most often rely on controlled inactivation of Pkd1 or Pkd2 using the Cre-loxP system. Our past work has extended beyond Cre-loxP to include modified bacterial artificial chromosome transgenics as well as a model in which second-hit inactivation occurs by a stochastic, Cre recombinase-independent process (Pkd2WS25). While loss of function models offer valuable information, we sought to determine whether ADPKD is actually reversible following Pkd gene reactivation, and if so, at what point in the course of ADPKD is reversal still possible. We developed mouse models that use adult inducible Cre–loxP for the initial Pkd gene inactivation and a separately inducible Flp–FRT system for subsequent reactivation of the same Pkd gene to address these questions. Applying this system to Pkd2, we found that cyst formation is rapidly reversible and that dilated cysts lined by proliferating, squamoid epithelial cells rapidly revert to non-proliferating columnar epithelia with normal appearing nephron lumens, accompanied by markedly decreased total kidney volume and preservation of kidney function. We will now determine the extent to which ADPKD is reversible by extending these studies to Pkd1 inactivation/reactivation and to the Pkd2WS25 mouse which does not require Cre and develops liver cysts as well. We will determine whether there is a minimum fraction of cyst cells that need to be targeted by reactivation to reverse ADPKD and determine the latest disease stage at which ADPKD retains reversibility. We will investigate cellular and molecular alterations operational during resolution of cysts including cell lineage analyses to trace the fates of specific cell types during the repair process, assess alterations in autophagic flux following PC re-expression as a possible modality for the changes in cell shape, and determine whether inflammation and fibrosis reverse with Pkd re-expression. We will attempt to model ADPKD repair in a cell culture-based system. Finally, we will determine the dynamic changes in transcriptionally defined in vivo cell populations during polycystin re-expression and reversal of ADPKD using single cell RNA sequencing (scRNA-seq). This will define the plasticity of the cell populations and the dynamic PC2-expression-dependent changes in transcriptional profiles leading to reversion from the cystic to a more normal nephron state. The reproducible and rapid initial time course of resolution of cysts affords a unique opportunity to monitor transitions within and between cell types in near real time and define on the scale of days the changes that are controlled by PC2 re-expression in the polycystic kidney environment in vivo. All of the findings from this study will be systematically correlated internally to provide an integrated cellular and molecular model for ADPKD repair and to define the capacity and trajectory of kidney repair possible in ADPKD.

常染色体显性多囊肾病 (ADPKD) 主要由 PKD1 和 PKD2 突变引起 PKD1 和 PKD2 分别编码多囊蛋白 1 (PC1) 和多囊蛋白 2 (PC2)。在了解多囊蛋白 (PC) 的功能方面取得了重大进展。基于体内直系同源基因小鼠模型，而该模型通常依赖于受控失活我们过去的工作已经扩展到 Cre-loxP 之外，包括修改后的 Pkd1 或 Pkd2。细菌人工染色体转基因以及通过细菌发生二次打击失活的模型随机的、独立于 Cre 重组酶的过程 (Pkd2WS25) 而功能损失模型提供了有价值的信息。信息，我们试图确定 Pkd 基因重新激活后 ADPKD 是否实际上是可逆的，以及如果是这样，ADPKD 过程中的什么阶段仍有可能逆转我们开发了使用成人的小鼠模型。用于初始 Pkd 基因失活的诱导型 Cre–loxP 和用于初始 Pkd 基因失活的单独诱导型 Flp–FRT 系统随后重新激活相同的 Pkd 基因来解决这些问题，我们将该系统应用于 Pkd2。发现囊肿的形成是快速可逆的，并且扩张的囊肿内衬有增殖的鳞状上皮细胞迅速恢复为非增殖的柱状上皮，具有正常的肾单位管腔，并伴有通过显着减少总肾体积和保留肾功能，我们现在将确定通过将这些研究扩展到 Pkd1 失活/重新激活以及 ADPKD 可逆的程度 Pkd2WS25小鼠不需要Cre并且也出现肝囊肿，我们将确定是否存在。是需要通过重新激活来逆转 ADPKD 并确定我们将研究 ADPKD 保留可逆性的最新疾病阶段的细胞和分子改变。在包囊解析过程中进行操作，包括细胞谱系分析，以追踪特定细胞类型的命运修复过程中，评估 PC 重新表达后自噬流的变化作为可能的修复方式细胞形状的变化，并确定炎症和纤维化是否随着 Pkd 重新表达而逆转。将尝试在基于细胞培养的系统中模拟 ADPKD 修复最后，我们将确定动态。在多囊蛋白重新表达和逆转过程中转录定义的体内细胞群发生变化 ADPKD 使用单细胞 RNA 测序 (scRNA-seq) 这将定义细胞群的可塑性和转录前导谱的动态 PC2 表达依赖性变化从囊性逆转囊肿消退的可重复且快速的初始时间过程提供了更正常的肾单位状态。近实时监测细胞类型内部和细胞类型之间的转变并按比例定义的独特机会体内多囊肾环境中 PC2 重新表达所控制的变化。这项研究的结果将在内部系统地关联起来，以提供综合的细胞和 ADPKD 修复的分子模型，并定义 ADPKD 肾脏修复的能力和轨迹。