Sharma Madhulika, Reif Gail A, Wallace Darren P
Department of Internal Medicine, The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS, United States; Department of Molecular and Integrative Physiology, The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS, United States.
Department of Internal Medicine, The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS, United States.
Methods Cell Biol. 2019;153:93-111. doi: 10.1016/bs.mcb.2019.05.008. Epub 2019 Jun 13.
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the relentless growth of numerous fluid-filled cysts in the kidneys. Mutations in PKD1 and PKD2, genes that encode polycystin 1 and 2, respectively, are responsible for most cases of ADPKD. Currently, the cellular mechanisms responsible for cyst formation remain poorly understood. In vitro models have been used by researchers to investigate cellular processes for cyst formation in carefully controlled experimental conditions. Madin-Darby canine kidney (MDCK) cells, a distal tubule epithelial cell line, were first used to form 3-dimensional (3-D) cysts within a hydrated collagen gel. This method was applied to epithelial cells cultured from cysts of human ADPKD kidneys, allowing investigators to study cellular mechanisms for cyst growth using cells that harbor the genetic mutations responsible for ADPKD in humans. Studies using ADPKD in vitro cysts have provided insight into cellular processes regulating cell proliferation, fluid secretion, and cell polarity. These assays were used to demonstrate the central role of cAMP agonists, such as arginine vasopressin, on cyst growth; and to test the effectiveness of potential therapeutic agents, including tolvaptan. Results obtained from in vitro cyst experiments demonstrate the translational value of cell model systems for investigating the mechanisms for cyst formation in human ADPKD. In this chapter, we describe protocols for growing ADPKD cells in a 3-D in vitro cyst assay and measuring total cyst volume by microscopy and image analysis.
常染色体显性多囊肾病（ADPKD）是一种常见的遗传性疾病，其特征是肾脏中无数充满液体的囊肿持续生长。PKD1和PKD2基因分别编码多囊蛋白1和多囊蛋白2，这两个基因的突变是大多数ADPKD病例的病因。目前，囊肿形成的细胞机制仍知之甚少。研究人员利用体外模型在精心控制的实验条件下研究囊肿形成的细胞过程。Madin-Darby犬肾（MDCK）细胞是一种远端肾小管上皮细胞系，最初被用于在水合胶原凝胶中形成三维（3-D）囊肿。该方法被应用于从人类ADPKD肾脏囊肿中培养的上皮细胞，使研究人员能够使用携带人类ADPKD相关基因突变的细胞来研究囊肿生长的细胞机制。使用ADPKD体外囊肿的研究为调节细胞增殖、液体分泌和细胞极性的细胞过程提供了见解。这些试验被用于证明环磷酸腺苷（cAMP）激动剂，如精氨酸加压素，在囊肿生长中的核心作用；并测试包括托伐普坦在内的潜在治疗药物的有效性。从体外囊肿实验获得的结果证明了细胞模型系统在研究人类ADPKD囊肿形成机制方面的转化价值。在本章中，我们描述了在三维体外囊肿试验中培养ADPKD细胞以及通过显微镜检查和图像分析测量囊肿总体积的方案。
