Polycystin Dependent Mechanisms of Tubular Plasticity

常染色体显性遗传性多囊肾病(ADPKD)主要由PKD1和PKD2突变引起 编码多囊蛋白-1(PC1)和多囊蛋白-2(PC2)。自从PKD1和PKD2被发现以来，有 在了解多囊蛋白(PC)的功能方面取得了重大进展。最近取得的许多进展 一直基于体内同源基因小鼠模型，而这种模型通常依赖于受控失活 使用Cre-loxP系统检测PKD1或PKD2的表达。我们过去的工作已经从CRE-loxP扩展到包括修改的 细菌人工染色体转基因以及一种模型，在该模型中，通过一种 随机、不依赖Cre重组酶的过程(Pkd2WS25)。虽然功能损失模型提供了有价值的 信息，我们试图确定在PKD基因重新激活后ADPKD是否真的是可逆的，并且 如果是这样的话，在ADPKD过程中的哪个时间点仍然有可能逆转。我们开发了使用成体的小鼠模型 用于初始PKD基因失活的可诱导Cre-loxP和单独可诱导的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PKD2系统，实现了对PKD2系统的实时监控 发现囊肿的形成是快速可逆的，扩张的囊肿内衬着增殖的鳞状上皮 细胞迅速恢复为非增殖性柱状上皮，伴有正常的肾单位管腔。 通过明显减少肾总体积，保存肾功能。我们现在将确定 通过将这些研究扩展到PKD1失活/再激活以及将这些研究扩展到 Pkd2WS25小鼠，不需要CRE，也会出现肝囊肿。我们将确定是否有 是需要通过重新激活来靶向以逆转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 在包囊溶解期间进行操作，包括进行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重新表达后自噬通量的变化作为一种可能的方式 细胞形态的变化，并决定炎症和纤维化是否随着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模拟ADPKD修复。最后，我们将确定动态 多囊蛋白再表达和逆转过程中体内转录定义的细胞群体的变化 ADPKD使用单细胞RNA测序(scRNA-seq)。这将定义细胞群体的可塑性和 PC2表达依赖的转录谱动态变化导致囊性癌逆转 恢复到更正常的肾单位状态。囊肿可重复且快速的初始时间进程提供了 独一无二的机会，可以近乎实时地监控单元格类型内部和之间的转换，并按比例定义 在体内的多囊肾环境中，PC2控制的变化的天数重新表达。所有的 这项研究的结果将进行系统的内部关联，以提供一个完整的细胞和 ADPKD修复的分子模型，并确定ADPKD可能的肾脏修复能力和轨迹。

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.