In Situ Reprogramming of Induced Nephron Progenitor Cells for Kidney Regeneration

描述(由申请人提供)： 这项建议描述了一种基于重新编程以改变细胞命运的最新进展的急性肾损伤的再生治疗的发展。具体地说，核重新编程技术将被用来创造一个诱导的肾单位祖细胞群体。肾脏的肾单位祖细胞是唯一已知的能够分化为肾单位所有部分的细胞类型。不幸的是，这些细胞只被发现 在帽间充质中，正常情况下成人肾脏中不存在。我们的合作者利特尔和他的同事已经确定了六种因素，可以将肾小管细胞转化为诱导的肾单位祖细胞。为了利用核重编程技术治疗急性肾损伤的最新进展，我们将创建一个诱导肾单位祖细胞的人工种群。这个种群将在体内将重编程基因输送到小鼠的肾脏细胞后在原位或就地创建。其他人使用原位重编程成功地治疗了糖尿病和心肌缺血的小鼠模型，这表明原位重编程技术可能也有助于保护肾脏免受缺血再灌注损伤。利用一种新开发的基因转移技术，重新编程的基因将被带入现有的肾脏细胞。可诱导的iggyBac转座子将用于将基因整合到肾细胞的基因组中，并将在多西环素诱导后启动重新编程。成人肾脏(SIX2，Cited1)中未发现的肾祖细胞的标志物将被检测，以确定在处理的肾脏中诱导的肾祖细胞的数量和位置。急性肾损伤后，将检测肾功能指标，如血肌酐和长期纤维化，以确定诱导的肾祖细胞群对损伤严重程度和恢复的影响。最后，我们将通过在双转基因小鼠模型中进行命运映射研究，确定诱导的肾单位祖细胞如何保护免受急性肾脏损伤。这些双转基因小鼠表达三苯氧胺诱导的Cited1启动子的Cre重组酶，并含有LacZ转基因基因，只有在Cre重组后才能表达LacZ。因此，只有Cited1诱导的肾小球祖细胞的后代细胞才会表达LacZ。这将允许通过可视化急性肾损伤后的所有LacZ细胞来追踪肾单位祖细胞的命运。我们推测，诱导的肾单位祖细胞可能通过迁移到受损部位并替换受损的细胞，从而恢复受损的肾单位，从而起到改善急性肾损伤的作用。这将代表一种从急性肾损伤中恢复的新机制。总之，原位重编程创造诱导的肾单位祖细胞是治疗急性肾损伤的一种潜在的有效方法。

DESCRIPTION (provided by applicant): This proposal describes the development of a regenerative treatment for acute kidney injury based on recent advances in reprogramming to change cell fate. Specifically, nuclear reprogramming technology will be employed to create a population of induced nephron progenitor cells. The nephron progenitor cells of the kidney are the only cell type known to be capable of differentiation into all parts of the nephron. Unfortunately, these cells are only found in the cap mesenchyme and are not normally present in the adult kidney. Our collaborators little and colleagues have identified six factors that can convert tubule cells into induced nephron progenitor cells. To capitalize on recent advances in nuclear reprogramming technology for the treatment of acute kidney injury, we will create an artificial population of induced nephron progenitors. This population will be created in situ, or in place, following the delivery of the reprogramming genes to kidney cells of mice in vivo. Others have used in situ reprogramming to successfully treat mouse models of diabetes and cardiac ischemia, suggesting that in situ reprogramming technology may also be useful for protection from ischemia reperfusion injury in the kidney. The reprogramming genes will be carried into the existing kidney cells using a newly developed gene transfer technique. An inducible piggyBac transposon will be used to integrate the genes into the genomes of renal cells and reprogramming will be initiated upon doxycycline induction. Markers of nephron progenitor cells that are not found normally in adult kidneys (Six2, Cited1) will be examined to determine the number and location of induced nephron progenitors in the treated kidneys. Following acute kidney injury, measures of kidney function such as serum creatinine and long-term fibrosis will be assayed to determine the effect of the induced nephron progenitor population on the severity and recovery from injury. Finally, we will determine how the induced nephron progenitor cells protect from acute kidney injury by performing fate- mapping studies in a double transgenic mouse model. These double transgenic mice express a tamoxifen- inducible Cre recombinase from the Cited1 promoter and contain a LacZ transgene that will express LacZ only after Cre recombination. Therefore, only cells that are progeny of the Cited1+ induced nephron progenitor cells will express LacZ. This will allow tracking of the nephron progenitor cell fate by visualization of all LacZ+ cells following acute kidney injury. We hypothesize that the induced nephron progenitors may function to ameliorate acute kidney injury by migrating to sites of damage and replacing the damaged cells, thereby restoring injured nephrons. This would represent a novel mechanism of recovery from acute kidney injury. In conclusion, in situ reprogramming to create induced nephron progenitors represents a potentially powerful treatment for acute kidney injury.