ENDOCYTOSIS IN CULTURED RENAL EPITHELIA

这些研究的长期目标是表征细胞中的内吞作用。 为了更好地了解内吞作用如何参与 在肾脏疾病过程中。 内吞作用是细胞的一项重要功能 生物学，在肾上皮细胞中起着特别重要的作用，因为它 参与排泄和再吸收的功能。 肾离子 转运部分受内吞插入和回收 转运蛋白，调节这种内吞运输的信号是 它们自身受激素的内吞作用调节。 小分子 小管滤液通过顶端胞吞作用以极快速度回收 在近端小管中，它因此对 肾小管滤液的内容物，由于摄取导致肾小管损伤 过滤的氨基糖苷类抗生素和过量或 蛋白尿中的有毒蛋白。 对成纤维细胞的研究已经建立了 定量荧光显微镜可用于表征 pH、融合可及性和配体积累的动力学， 在核内体中分类。 细胞内吞动力学已阐明 内吞分选的机制和内体pH值的测量 确定了核内体酸化在 溶酶体水解，病毒和毒素渗透到胞质溶胶中， 细胞内药物隔离。 尽管这些过程对于 肾上皮细胞的正常和病理功能，比较研究 由于技术原因，尚未在极化上皮细胞中进行 困难 随着改进的光学仪器的发展， 适当的数字图像处理技术和转染的模型细胞 系统，这些上皮细胞的研究现在是可行的。 具体地说， 拟议的研究将首先解决内体pH的调节， 模型肾上皮。 一个完整而具体的特征 内体在循环、降解和转胞吞途径中的作用 将从顶侧和基底侧质膜域进行 使用活细胞的荧光比率共聚焦显微镜。 的 内吞分选机制也将解决，使用类似的 定量共聚焦显微镜技术。 特定类型的 内体可以根据其内容物进行功能鉴定， 不同配体在细胞中积累和流出的动力学 这些内体可以阐明内吞分选的特定步骤。 调节或干扰内体的生物相关因子 还将研究酸化和内吞运输。 这些 研究阐述了内吞作用的基本过程， 对非极化细胞的正常细胞功能至关重要，但 在上皮细胞中进行了研究。 本报告提供的信息 这项研究对上皮细胞生物学家和 肾生物学家寻求了解生理和病理肾 细胞过程

The long term objective of these studies is to characterize endocytosis in renal epithelia in order to better understand how endocytosis participates in renal disease processes. Endocytosis is an important function in cell biology, and in kidney epithelia plays a particularly important role as it participates in the functions of excretion and resorption. Renal ion transport is partially regulated by endocytic insertion and retrieval of transporters, and the signals regulating this endocytic traffic are themselves regulated by endocytosis of hormones. Small macromolecules in the tubule filtrate are reclaimed via furious rates of apical endocytosis in the proximal tubule which is consequently particularly sensitive to the contents of the tubule filtrate, with tubular damage resulting from uptake of filtered aminoglycoside antibiotics and from the uptake of excessive or toxic proteins during proteinuria. Studies of fibroblasts have established that quantitative fluorescence microscopy may be used to characterize the pH, fusion accessibility and the kinetics of ligand accumulation and sorting in endosomes. The intracellular endocytic kinetics have elucidated mechanisms of endocytic sorting and measurements of endosome pH have established the role of endosome acidification in the processes of lysosomal hydrolysis, virus and toxin penetration into the cytosol and intracellular drug sequestration. Although these processes are critical to normal and pathological function of renal epithelia, comparable studies have not yet been conducted in polarized epithelial cells due to technical difficulties. With the development of improved optical instrumentation, appropriate digital image processing techniques and transfected model cell systems, these studies of epithelial cells are now feasible. Specifically, the proposed studies will first address the regulation of endosome pH in model renal epithelia. A complete and specific characterization of endosomes on the recycling, degradative and transcytotic pathways derived from the apical and basolateral plasma membrane domains will be conducted using fluorescence ratio confocal microscopy of living cells. The mechanisms of endocytic sorting will also be addressed, using similar techniques of quantitative confocal microscopy. Particular types of endosomes can be functionally identified on the basis of their contents and the kinetics with which different ligands accumulate in, and efflux from these endosomes can elucidate specific steps of endocytic sorting. Biologically relevant factors regulating or perturbing endosome acidification and endocytic trafficking will also be studied. These studies address fundamental processes of endocytosis which have proven to be critical to proper cell function in non-polarized cells, but have not been studied in epithelial cells. The information provided by this research will be valuable to both epithelial cell biologists and also to renal biologists seeking to understand physiological and pathological renal cellular processes.