Ferrell Nicholas, Sandoval Ruben M, Molitoris Bruce A, Brakeman Paul, Roy Shuvo, Fissell William H
Department of Medicine, Division of Nephrology, Vanderbilt University Medical Center, Nashville, TN, United States; Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States.
Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States.
Methods Cell Biol. 2019;153:43-67. doi: 10.1016/bs.mcb.2019.04.010. Epub 2019 May 24.
Renal tubular epithelial cells are consistently exposed to flow of glomerular filtrate that creates fluid shear stress at the apical cell surface. This biophysical stimulus regulates several critical renal epithelial cell functions, including transport, protein uptake, and barrier function. Defining the in vivo mechanical conditions in the kidney tubule is important for accurately recapitulating these conditions in vitro. Here we provide a summary of the fluid flow conditions in the kidney and how this translates into different levels of fluid shear stress down the length of the nephron. A detailed method is provided for measuring fluid flow in the proximal tubule by intravital microscopy. Devices to mimic in vivo fluid shear stress for in vitro studies are discussed, and we present two methods for culture and analysis of renal tubule epithelial cells exposed physiological levels of fluid shear stress. The first is a microfluidic device that permits application of controlled shear stress to cells cultured on porous membranes. The second is culture of renal tubule cells on an orbital shaker. Each method has advantages and disadvantages that should be considered in the context of the specific experimental objectives.
肾小管上皮细胞持续暴露于肾小球滤液的流动中，这种流动在细胞顶端表面产生流体剪切力。这种生物物理刺激调节着几种关键的肾上皮细胞功能，包括转运、蛋白质摄取和屏障功能。明确肾小管内的体内力学条件对于在体外准确重现这些条件很重要。在这里，我们总结了肾脏中的流体流动条件，以及这如何转化为沿肾单位长度不同水平的流体剪切力。提供了一种通过活体显微镜测量近端小管中流体流动的详细方法。讨论了用于体外研究模拟体内流体剪切力的装置，并且我们展示了两种培养和分析暴露于生理水平流体剪切力的肾小管上皮细胞的方法。第一种是微流控装置，它允许对培养在多孔膜上的细胞施加可控的剪切力。第二种是在轨道振荡器上培养肾小管细胞。每种方法都有优点和缺点，应根据具体实验目标加以考虑。
