Synthetic approaches to study cell polarity related kidney defects

研究细胞极性相关肾脏缺陷的综合方法

• 批准号: 10521239
• 负责人: Louis Skjei Prahl
• 金额: $ 7.38万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10521239
• 关键词: 3-Dimensional; Address; Adult; Affect; Automobile Driving; Behavior; Biochemical; Blood Circulation; Cell Communication; Cell Polarity; Cells; Child; Childhood; Chronic Kidney Failure; Complex; Congenital Abnormality; Cues; DNA; Defect; Development; Developmental Process; Diameter; Dimensions; Disease; Duct (organ) structure; Dysplasia; Embryo; End stage renal failure; Engineering; Epithelial Cells; Epithelium; Failure; Fatty acid glycerol esters; Fluorescence; Future; Genes; Genetic; Goals; Growth; Health; Human; Kidney; Kidney Diseases; Knock-out; Life; Ligands; Logic; Loose connective tissue; Lung; Measures; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Modeling; Molecular Genetics; Morphology; Movement; Mus; Mutation; Neuroglia; Organoids; Outcome; Pattern; Phosphotransferases; Physiological; Population; Positioning Attribute; Process; Production; Proteins; Protocols documentation; Renal tubule structure; Reporter; Role; Shapes; Signal Pathway; Signal Transduction; Standardization; Structure; Syndrome; System; Technology; Testing; Time; Tissue Engineering; Tissues; Trees; Tube; Ureter; Urinary tract; Urine; Waste Products; cell type; collecting tubule structure; congenital anomalies of the kidney; genetic approach; high-throughput drug screening; human disease; human model; improved; in vivo; induced pluripotent stem cell; kidney cell; mathematical model; nephrogenesis; neurotrophic factor; novel therapeutics; planar cell polarity; prevent; response; tissue support frame; tool

ABSTRACT Congenital abnormalities of the kidney and urinary tract (CAKUT) account for ~50% of childhood chronic kidney disease cases. Many CAKUT defects involve imprecise sizing or spacing of kidney collecting duct structures, such as duplicated ureters, cystic collecting ducts, and renal hypoplasia, which can lead to severe health problems including end-stage kidney disease. As such, there is a critical need to understand how developmental processes promote proper sizing, spacing, and positioning of kidney structures, so that CAKUT defects can be corrected. Kidney development begins through tree-like outgrowth of the ureteric bud epithelium (the future collecting duct network) into a loose connective tissue or mesenchyme. Precisely positioning ureteric bud tubules within this network requires tight control of Ret kinase signaling. Ret responds to secreted glial cell-derived neurotrophic factor (GDNF) from surrounding mesenchymal cells and mutations that affect Ret-GDNF signaling cause CAKUT defects. While Ret-GDNF signaling drives the proliferative expansion of ureteric bud epithelial cells, emerging evidence suggests that “planar cell polarity” (PCP) – a mechanism by which cells sense their planar positions within sheets and tubes – controls the shape of the collecting duct network. Significantly, recent findings indicate that mesenchymal cells expressing the PCP genes Fat4 and Dchs1 prevent improper ureteric bud branching and mutations in these genes cause CAKUT defects in mice and humans. However, it is unclear how interfaces between PCP-expressing mesenchymal cells and Ret-expressing epithelial cells enforce the precise sizing and spacing of collecting duct tubules and avoid defects. The objective of this proposal is to create controlled spatial interfaces between PCP-expressing cells and Ret-expressing cells and study their impact on Ret-GDNF signaling levels and the resultant effect on the size and shape of epithelial structures. Aim 1 of this proposal establishes a DNA-based cell patterning technology that enables the production of cell interfaces in engineered tissues. This cell patterning technology will be used to create interfaces between PCP-expressing cells and Ret-expressing cells. Cells expressing fluorescence-based kinase activity reporters and mathematical modeling will be used to study how these spatial interfaces influence Ret-GDNF signaling. Aim 2 of this proposal will pattern the two cell types in 3D tissue scaffolds and study the effects of interfaces on the size and shape of resulting epithelial structures. The central hypothesis of this proposal is that PCP-expressing cells locally restrict Ret-GDNF-driven epithelial tissue growth at interfaces, thereby producing structures of defined size and shape. Together, the approaches developed in this proposal will improve our understanding of the cellular mechanisms that cause CAKUT defects and create new tools to build defined tissue structures in organoid models of human disease.

抽象的 肾脏和尿路（Cakut）的先天性异常（Cakut）约占儿童慢性的50％ 肾脏疾病病例。许多Cakut缺陷涉及肾脏收集管道的尺寸或间距 结构，例如重复的输尿管，囊性收集导管和肾功能低下，这可能导致严重 健康问题，包括末期肾脏疾病。因此，迫切需要了解如何 发展过程促进肾脏结构的适当尺寸，间距和定位，以便cakut 可以纠正缺陷。肾脏发育始于输尿管芽的类似树状的产物 上皮（未来的收集管网）进入松散的结缔组织或间质。恰恰 在该网络中定位输尿管芽管需要严格控制RET激酶信号。 RET响应 来自周围间充质细胞和突变的分泌神经胶质细胞衍生的神经营养因子（GDNF） 影响RET-GDNF信号传导会导致Cakut缺陷。而RET-GDNF信号传导驱动增殖 输尿管芽上皮细胞的膨胀，新出现的证据表明“平面细胞极性”（PCP） - A 细胞感知其平面位置在板和管中的机制 - 控制 收集管道网络。值得注意的是，最近的发现表明表达PCP的间充质细胞 FAT4和DCHS1基因可防止这些基因的输尿管芽分支不当，引起Cakut 老鼠和人类的缺陷。但是，目前尚不清楚如何表达PCP的间充质之间 细胞和表达表达的上皮细胞可实施收集管管的精确尺寸和间距 避免缺陷。该提案的目的是在表达PCP之间创建受控的空间接口 细胞和表达细胞的细胞，并研究其对RET-GDNF信号水平的影响，并对结果对 上皮结构的大小和形状。该提案的目标1建立了基于DNA的细胞模式 能够生产工程组织中细胞界面的技术。这种细胞图案技术 将用于在表达PCP的细胞和表达Ret表达细胞之间创建接口。表达细胞 基于荧光的激酶活性记者和数学建模将用于研究这些 空间接口会影响RET-GDNF信号传导。该提案的目标2将在3D中对两种单元格类型进行模式 组织支架并研究界面对所得上皮结构大小和形状的影响。 该提案的中心假设是表达PCP的细胞局部限制了RET-GDNF驱动的上皮 界面的组织生长，从而产生定义大小和形状的结构。在一起，方法 在此提案中开发的将提高我们对导致Cakut的细胞机制的理解 缺陷并创建新工具，以在人类疾病的器官模型中构建定义的组织结构。