Polycystin Dependent Mechanisms of Tubular Plasticity

管状可塑性的多囊蛋白依赖性机制

• 批准号: 10427385
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427385
• 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; Gold; 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; Sum; System; Testing; Therapeutic; Time; Tissues; Transgenic Organisms; Transplantation; Tubular formation; Validation; Work; base; cell type; field study; human disease; in vivo; 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脏疾病（ADPKD）由PKD1和PKD2突变产生 分别编码polcysytin-1（PC1）和polycystin-2（PC2）。由于发现了PKD1和PKD2，因此 在理解多碳素（PC）的功能方面，已经取得了重大进展。最近的进步已有很多 是基于体内直系同源基因模型的基础 使用Cre-loxp系统的PKD1或PKD2的of。我们过去的工作已经超越了Cre-loxp，包括修改 细菌人造染色体转化以及第二次灭活的模型。 随机，CRE重组酶独立的过程（PKD2WS25）。功能模型的损失提供价值 信息，我们感觉确定PKD基因重新激活后ADPKD是否实际上是可逆的，并且 如果是这样，在ADPKD过程中的什么时候仍然有可能。我们开发了使用成人的鼠标模型 初始PKD基因失活的诱导型Cre -loxP和单独诱导的FLP – FRT系统 随后对同一PKD基因的重新激活以解决这些问题。将此系统应用于PKD2，我们 发现囊肿的形成迅速可逆，并且通过增殖，鳞状上皮衬里的囊肿扩张 细胞迅速恢复为正常出现的肾单位的非增殖柱状上皮，完成 明显减少了肾脏总量和肾功能的保存。现在我们将确定 通过将这些研究扩展到PKD1灭活/重新激活以及向ADPKD可逆的程度可逆的程度 PKD2WS25小鼠不需要CRE并也会发展出肝囊肿。我们将确定是否在那里 是囊肿细胞的最小比例，需要通过重新激活来逆转ADPKD并确定 ADPKD保留可逆性的最新疾病阶段。我们将研究细胞和分子改变 在分辨出包括细胞谱系分析的囊肿的过程中进行操作，以追踪特定细胞类型的命运 修复过程，PC重新表达后自噬通量的评估改变是可能的方式 细胞形状的变化，并确定炎症和纤维化是否会随着PKD的重新表达而逆转。我们 将尝试在基于细胞培养的系统中对ADPKD修复进行建模。最后，我们将确定动态 在多囊这重新表达和逆转期间，在体内细胞群体中的转录定义的变化 使用单细胞RNA测序（SCRNA-SEQ）的ADPKD。这将定义细胞种群的可塑性和 转录曲线的动态PC2表达依赖性变化，导致与囊性相反 达到更正常的肾单状态。可再现和快速的初始时间疗程的囊肿分辨率提供了 独特的机会来监视单元类型内和几乎实时之间的过渡，并在规模上定义 几天，在体内多囊肾脏环境中PC2重新表达的变化。所有人 这项研究的发现将在内部系统地相关联，以提供综合的细胞和 用于ADPKD修复的分子模型，并定义ADPKD中可能的肾脏修复的能力和轨迹。