Polycystin Dependent Mechanisms of Tubular Plasticity

管状可塑性的多囊蛋白依赖性机制

• 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
• 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

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PKD 1和PKD 2突变引起 分别编码多囊蛋白-1（PC 1）和多囊蛋白-2（PC 2）。自从PKD 1和PKD 2被发现以来， 对多囊蛋白（polycystins，PC）功能的研究取得了重大进展。最近的许多进展 基于体内正向基因小鼠模型，其通常依赖于受控失活 Pkd 1或Pkd 2的使用Cre-loxP系统。我们过去的工作已经扩展到Cre-loxP之外， 细菌人工染色体转基因以及其中通过细菌人工染色体转基因发生二次打击失活的模型。 随机，Cre重组酶独立过程（Pkd 2 WS 25）。虽然功能丧失模型提供了宝贵的 信息，我们试图确定是否ADPKD实际上是可逆的Pkd基因重新激活后， 如果是这样，在ADPKD的过程中，在什么时候逆转仍然是可能的。我们开发了小鼠模型， 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。将该系统应用于Pkd 2，我们 发现囊肿的形成是迅速可逆的，扩张的囊肿内衬着增殖的鳞状上皮细胞， 细胞迅速恢复为非增殖性柱状上皮细胞，肾单位管腔正常， 通过显著减少肾脏总体积和保护肾功能。我们现在将决定 通过将这些研究扩展到Pkd 1失活/再活化和 Pkd 2 WS 25小鼠，其不需要Cre并且也发展肝囊肿。我们将确定是否有 是需要通过再活化靶向以逆转ADPKD并确定 ADPKD保持可逆性的最新疾病阶段。我们将研究细胞和分子的变化 包括细胞谱系分析，以追踪特定细胞类型的命运 修复过程，评估PC再表达后自噬通量的改变作为一种可能的方式， 细胞形态的变化，并确定炎症和纤维化是否逆转与Pkd的重新表达。我们 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 在多囊蛋白再表达和逆转多囊蛋白表达过程中转录确定的体内细胞群的变化 使用单细胞RNA测序（scRNA-seq）检测ADPKD。这将定义细胞群体的可塑性， 转录谱中的动态PC 2表达依赖性变化导致从囊性变的逆转， 恢复到正常的肾单位状态囊肿消退的可重复和快速的初始时间过程提供了 在接近真实的时间内监测细胞类型内和细胞类型之间的转换并在规模上定义的独特机会 在多囊肾的体内环境中，由PC 2再表达控制的变化。所有 这项研究的结果将被系统地内部关联，以提供一个综合的细胞和 ADPKD修复的分子模型，并确定ADPKD可能的肾脏修复能力和轨迹。