In Situ Reprogramming of Induced Nephron Progenitor Cells for Kidney Regeneration

诱导肾单位祖细胞原位重编程促进肾脏再生

• 批准号: 10064117
• 负责人: Lauren Elizabeth Woodard
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064117
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Adult; Affect; Beta Cell; Biological Assay; Biological Markers; Birth; Cardiac Myocytes; Categories; Cell Differentiation process; Cells; Collaborations; Creatinine; Development; Diabetic mouse; Dialysis procedure; Disease; Doxycycline; End stage renal failure; Enterobacteria phage P1 Cre recombinase; Environment; Epithelial; Epithelium; Exocrine pancreas; Fibroblasts; Fibrosis; Foundations; Gene Transfer Techniques; Genes; Genetic Recombination; Genome; Goals; Growth Factor; In Situ; Injections; Injury to Kidney; Insulin; Intensive Care Units; Ischemia; Kidney; Kidney Diseases; Kidney Transplantation; LacZ Genes; Length; Location; Measures; Mediating; Mesenchymal Stem Cell Transplantation; Mesenchyme; Methods; Morbidity - disease rate; Mus; Myocardial Ischemia; Natural regeneration; Nephrons; Neurons; Patient Care; Patients; Plasmids; Pluripotent Stem Cells; Population; Process; Recovery; Renal Replacement Therapy; Renal function; Renal pelvis; Renal tubule structure; Reperfusion Injury; Reperfusion Therapy; Research; Serum; Severities; Site; Stains; Supportive care; Tamoxifen; Techniques; Technology; Time; Transgenes; Transgenic Mice; Transplantation; Tubular formation; United States; Veterans; Visualization; base; cell dedifferentiation; cell injury; cell type; cost; design; in vivo; injured; injury recovery; insight; kidney cell; mortality; mouse model; novel; nuclear reprogramming; paracrine; preclinical development; prevent; progenitor; promoter; public health relevance; regenerative; repaired; response; screening; stem cell therapy; stem cells; targeted treatment; therapy design; transcription factor

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.

描述（由申请人提供）： 该提案描述了基于重新编程以改变细胞命运的最新进展的急性肾损伤再生治疗的发展。具体而言，将采用核重编程技术来产生诱导的肾单位祖细胞群。肾的肾单位祖细胞是已知能够分化成肾单位的所有部分的唯一细胞类型。不幸的是，这些细胞只在 在帽间充质中，通常不存在于成人肾脏中。我们的合作者little和同事已经确定了六个可以将肾小管细胞转化为诱导肾单位祖细胞的因子。为了利用核重编程技术治疗急性肾损伤的最新进展，我们将建立一个人工诱导的肾单位祖细胞群。在体内将重编程基因递送至小鼠的肾细胞后，将原位或原位产生该群体。其他人已经使用原位重编程成功地治疗糖尿病和心脏缺血的小鼠模型，这表明原位重编程技术也可以用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，重编程基因将被携带到现有的肾细胞中。诱导型piggyBac转座子将用于将基因整合到肾细胞的基因组中，并且在多西环素诱导后将启动重编程。将检查成人肾脏中正常未发现的肾单位祖细胞标记物（Six 2，Cited 1），以确定治疗肾脏中诱导肾单位祖细胞的数量和位置。在急性肾损伤后，将测定肾功能的测量，如血清肌酐和长期纤维化，以确定诱导的肾单位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定位研究来确定诱导的肾单位祖细胞如何保护急性肾损伤。这些双转基因小鼠从Cited1启动子表达他莫昔芬诱导型Cre重组酶，并含有仅在Cre重组后表达LacZ的LacZ转基因.因此，只有Cited1+诱导的肾单位祖细胞的后代细胞才会表达LacZ。这将允许在急性肾损伤后通过可视化所有LacZ+细胞来跟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损伤部位并取代受损细胞，从而恢复受损的肾单位，从而改善急性肾损伤。这将代表急性肾损伤恢复的新机制。总之，原位重编程以产生诱导的肾单位祖细胞代表了急性肾损伤的潜在有力治疗。