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 先天性肾脏和泌尿道异常 (CAKUT) 约占儿童慢性病的 50% 肾脏疾病病例。许多 CAKUT 缺陷涉及肾集合管的尺寸或间距不精确 结构，如重复的输尿管、囊性集合管和肾发育不全，这可能导致严重的 健康问题，包括终末期肾病。因此，迫切需要了解如何 发育过程促进肾脏结构的适当大小、间距和定位，以便 CAKUT 缺陷可以被纠正。肾脏发育是通过输尿管芽的树状生长开始的 上皮（未来的集合管网络）转变为疏松结缔组织或间质。恰恰 将输尿管芽小管定位在该网络内需要严格控制 Ret 激酶信号传导。雷特回应 从周围的间充质细胞分泌胶质细胞源性神经营养因子（GDNF）和突变 影响 Ret-GDNF 信号传导的因素会导致 CAKUT 缺陷。虽然 Ret-GDNF 信号传导驱动增殖 随着输尿管芽上皮细胞的扩张，新出现的证据表明“平面细胞极性”（PCP）—— 细胞感知其在片材和管内的平面位置的机制 - 控制形状 收集管网络。值得注意的是，最近的研究结果表明表达 PCP 的间充质细胞 Fat4 和 Dchs1 基因可防止输尿管芽分支不当，这些基因的突变会导致 CAKUT 小鼠和人类的缺陷。然而，尚不清楚表达 PCP 的间充质细胞之间如何相互作用。 细胞和表达 Ret 的上皮细胞强制集合管小管的精确尺寸和间距， 避免缺陷。该提案的目标是在 PCP 表达之间创建受控的空间界面 细胞和 Ret 表达细胞，并研究它们对 Ret-GDNF 信号水平的影响以及由此产生的影响 上皮结构的大小和形状。该提案的目标 1 建立基于 DNA 的细胞模式 能够在工程组织中产生细胞界面的技术。这种细胞图案化技术 将用于在表达 PCP 的细胞和表达 Ret 的细胞之间创建界面。细胞表达 基于荧光的激酶活性报告器和数学模型将用于研究这些 空间界面影响 Ret-GDNF 信号传导。该提案的目标 2 将在 3D 中对两种细胞类型进行图案化 组织支架并研究界面对所得上皮结构的尺寸和形状的影响。这 该提议的中心假设是 PCP 表达细胞局部限制 Ret-GDNF 驱动的上皮细胞 组织在界面处生长，从而产生限定尺寸和形状的结构。共同采取的方法 该提案中开发的内容将提高我们对导致 CAKUT 的细胞机制的理解 缺陷并创建新工具来在人类疾病的类器官模型中构建明确的组织结构。