PDF(Pubmed)
Abstract：
Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi ligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is > 5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.
摘要：
近端小管（PT）细胞维持高容量的顶端胞吞途径，以回收基本上所有逃脱肾小球滤过屏障的蛋白质。多配体受体megalin和cubilin在PT细胞中正常过滤蛋白的内吞摄取中起关键作用，但也有助于肾毒性药物的摄取。包括氨基糖苷类.我们先前证明，在连续流体剪切应力（FSS）下培养的负鼠肾（OK）细胞在体内重现PT细胞的基本功能特性方面优于在静态条件下培养的细胞。为了识别高容量的驱动因素，PT中有效的内吞途径，我们比较了FSS培养的OK细胞和具有较低内吞活性的静态培养的OK细胞。Megalin和cubilin表达增加，与在静态条件下培养的细胞相比，FSS培养的细胞中白蛋白的内吞摄取>5倍。为了了解受体表达的差异，分布，贩运率有助于增加吸收，我们使用了生化，形态学,和数学建模方法来比较FSS和静态培养的OK细胞中的megalin流量。我们的模型预测，在FSS下培养细胞会增加megalin运输中所有步骤的速率。重要的是,这个模型解释了为什么，尽管看似违反直觉的观察（细胞表面的megalin含量减少，与溶酶体的共定位更高，和FSS培养细胞中表面标记的巨蛋白的半衰期较短），与静态生长的细胞相比，白蛋白的摄取显着增加。我们还表明，与静态生长的细胞相比，FSS培养的OK细胞更准确地表现出介导体内肾毒性药物摄取的机制。因此，这种培养模型提供了一个有用的平台来理解药物摄取机制，对发展肾毒性损伤预防干预措施具有重要意义。
