Establishing and Mimicking Patterning Mechanisms in the Distal Nephron Tubule and Kidney Organoid

建立和模拟远端肾小管和肾类器官的模式机制

• 批准号: 10719178
• 负责人: Nils Olof Lindstrom
• 金额: $ 65.02万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10719178
• 关键词: Address; Adopted; Adult; Automobile Driving; Birth; Bladder; Blood Pressure; Blood flow; Body Fluids; Cell Differentiation process; Cell Lineage; Cell Separation; Cells; Childhood; Chromosome Mapping; Clinical; Confusion; Congenital Abnormality; Cues; DNA; Data; Defect; Development; Developmental Anatomy; Dialysis procedure; Disease model; Distal; Dose; Drainage procedure; Embryo; End stage renal failure; Ensure; Etiology; Excretory function; Fluid Balance; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Markers; Genetic Transcription; Goals; Human; In Vitro; Infant; Kidney; Kidney Diseases; Life; Life Expectancy; Ligands; Link; Maps; Mediating; Molecular; Mouse Strains; Mus; Mutate; Mutation; Nephrology; Nephrons; Newborn Infant; Organ Transplantation; Organoids; Patients; Pattern; Physiological; Physiology; Population; Production; Proteins; Protocols documentation; Publishing; Regulation; Regulator Genes; Renal function; Replacement Therapy; Running; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Sodium Chloride; Specific qualifier value; System; Techniques; Testing; Therapeutic; Time; Transcriptional Regulation; Urinary tract; Urine; Validation; Variant; Water; Work; absorption; beta catenin; blood filtration; blood pressure control; cell replacement therapy; cell type; congenital anomalies of the kidney; diagnostic tool; effective intervention; experimental study; fetal; human stem cells; improved; in vitro Model; in vivo; induced pluripotent stem cell; insight; molecular modeling; mortality; nephrogenesis; novel; novel therapeutics; practical application; programs; regenerative therapy; solute; stem cell model; stem cells; synthetic biology; tool; transcription factor

During embryonic and fetal stages, the kidneys develop millions of nephrons that generate highly specialized cells. These cells ensure that blood flowing into the kidney is filtered and required substances are reabsorbed while unwanted metabolites and solutes are led to the bladder for excretion. Birth defects are common in the kidney, ~ 1/100 of all births have a so-called Congenital Anomaly of the Kidney and Urinary Tract (CAKUT). At the most severe end of CAKUT, newborns are missing kidney functionality, and their life expectancy is less than one year. Most abnormalities have no current effective interventions and genetic changes lack context. There is thus a critical need to understand where developmental defects arise and to generate new therapies restoring or replacing kidney function. In our work we have used single cell omics and molecular characterizations of human and mouse kidneys to provide a blueprint for how nephrons form and maps for to replicate this in human stem cell-derived kidney organoids. In doing so we provide a genetic and developmental context to genes identified in CAKUT patients. In this proposal we will follow these leads and address three outstanding questions in developmental nephrology. In Aim 1, we investigate the embryonic origins of distal nephron tubule segments. We will perform the first single cell omic analysis linking developing and adult kidneys. This provides a roadmap for how cells differentiate. We will use new genetic mouse lineage-tracing tools to test how cells in the early distal nephron relate to functional cells in mature kidneys. These experiments will map where genes are required as the nephron develops. In Aim 2, we will investigate how proteins that turn genes on and off control the development of the distal nephron. We will use a technique called Cut&Run to analyze how genes often mutated in CAKUT, control DNA and gene expression. We will also activate signaling pathways and alter the expression of genes linked to CAKUT. This will allow us to directly study how distal nephron cells form provide causality between gene expression and regulation. We will use our new system to generate hundreds of nephrons from human stem cells in - synchronized nephroids. In this system, nephrons develop at the same time and pace, unlike in the body where nephrons from many developmental stages form near each other. Our system provides a unique advantage to study, manipulate, and isolate cells from nephrons at the same developmental stage. The data we collect will show how genes are activated. In Aim 3 we address a fundamental question in developmental nephrology - how is the nephron initially patterned? To do this we will use synthetic cellular organizers that secrete signaling proteins to pattern our synchronized nephroids. We will study how signal ligands control nephron formation and patterning. This also has a practical application as we can gain control over nephroid patterning. Our system will inform our efforts to build massive parallel arrays of nephroids for replacement therapies and disease modeling. A strength of the proposal is the unique expertise intersecting human and mouse kidney genetics, a novel system of stem cell derived kidney organoids, and synthetic biology.

在胚胎和胎儿阶段，肾脏发育成数百万个肾单位， 细胞这些细胞确保流入肾脏的血液被过滤，所需物质被重吸收 而不需要的代谢物和溶质被引导到膀胱排泄。出生缺陷是常见的， 在所有出生的婴儿中，约1/100的婴儿患有所谓的先天性肾脏和泌尿道异常（CAKUT）。在 CAKUT最严重的结局是，新生儿失去了肾脏功能，他们的预期寿命不到 一年大多数异常目前没有有效的干预措施，遗传变化缺乏背景。有 因此，迫切需要了解发育缺陷出现的地方，并产生新的治疗方法， 或替代肾功能。在我们的工作中，我们使用了单细胞组学和分子表征， 人类和小鼠肾脏，以提供肾单位如何形成和映射的蓝图，从而在人类中复制这一点 干细胞衍生的肾类器官。这样我们就为基因提供了一个遗传和发育的背景 在CAKUT患者中发现。在本提案中，我们将遵循这些线索，解决三个悬而未决的问题 发展性肾病学目的1：研究远侧肾单位小管段的胚胎起源。 我们将进行第一个单细胞组学分析，将发育中的肾脏和成人肾脏联系起来。这提供了路线图 细胞是如何分化的。我们将使用新的遗传小鼠谱系追踪工具来测试早期远端的细胞是如何在小鼠体内生长的。 肾单位与成熟肾脏中的功能细胞有关。这些实验将绘制基因所需的位置， 肾单位发育。在目标2中，我们将研究如何打开和关闭基因的蛋白质控制的蛋白质。 远端肾单位的发育。我们将使用一种名为“切与跑”的技术来分析基因如何经常突变， 在CAKUT中，控制DNA和基因表达。我们还将激活信号通路， 与CAKUT相关的基因。这将使我们能够直接研究远端肾单位细胞如何形成提供因果关系 基因表达和调控之间的联系我们将用我们的新系统从 人类干细胞在同步化的类肾中。在这个系统中，肾单位以相同的时间和速度发育， 不像在体内，来自许多发育阶段的肾单位彼此靠近形成。我们的系统提供 这对于研究、操作和分离处于相同发育阶段的肾单位的细胞具有独特的优势。的 我们收集的数据将显示基因是如何被激活的。在目标3中，我们解决了发展中的一个基本问题。 肾脏学-肾单位最初是如何形成的？为了做到这一点，我们将使用合成细胞组织者， 分泌信号蛋白来构建我们的同步类肾。我们将研究信号配体如何控制 肾单位的形成和图案化。这也有实际应用，因为我们可以控制肾线， 模式化我们的系统将为我们建造大规模并行肾阵列的努力提供信息， 治疗和疾病建模。该提案的一个优点是独特的专业知识， 小鼠肾脏遗传学，干细胞衍生的肾脏类器官的新系统，以及合成生物学。