Maine Medical Center Research Institute, Clinical and Translational Research, Portland, Maine 04102, USA.
Oxid Med Cell Longev. 2008 Oct-Dec;1(1):33-8. doi: 10.4161/oxim.1.1.6491.
We have developed and evaluated an in vitro culture method for assessing ischemic injury in primary mouse renal tubular epithelial cells (RTEC) in which to explore the pathobiology underlying acute kidney injury. RTEC were predominately of proximal tubule origin which is most susceptible to ischemic injury as compared to other nephron segments. Oxidative stress was induced by chemically depleting ATP using Antimycin A and 2-Deoxy-D-Glucose and by exposing cells to a 1% oxygen environment. Necrotic injury was assessed by measuring LDH released into culture supernatants. Optimal dose and time of exposure to each injury agent was determined for induction of mild, moderate and severe ischemic injury defined as LDH release of </= 20%, 21-49% and >/= 50% above baseline respectively. Antimycin A and 2-Deoxy-D-Glucose produced a progressive increase in LDH release which was time dependent but chemical concentration independent. A 1% oxygen environment also induced cell injury over time but only if glucose was absent from the culture media. Antimycin A was most effective at inducing oxidative stress causing a mean LDH release of 61% at 48 hr compared to 19% and 50% LDH release induced by 2-Deoxy-D-Glucose and by exposure to 1% oxygen respectively at the same 48 hour time point.The cell culture method described provides several advantages including the use of serum free media and the ability to grow primary cells without matrix support. The LDH assay for injury assessment is reproducible, cost effective, objective and minimizes background cell death. A simple method for the culture and injury of primary mouse renal tubular epithelial cells has thereby been established and provides a useful tool for future investigations of ischemic kidney injury.
我们已经开发并评估了一种体外培养方法，用于评估原代小鼠肾小管上皮细胞（RTEC）中的缺血性损伤，以探索急性肾损伤的病理生物学。与其他肾单位节段相比，RTEC 主要来自近端肾小管，对缺血性损伤最敏感。通过使用 Antimycin A 和 2-脱氧-D-葡萄糖化学耗尽 ATP 以及将细胞暴露于 1%氧气环境来诱导氧化应激。通过测量培养上清液中释放的 LDH 来评估坏死性损伤。确定了每种损伤剂的最佳剂量和暴露时间，以诱导轻度、中度和重度缺血性损伤，定义为 LDH 释放分别为基线以上 </= 20%、21-49%和 >/= 50%。Antimycin A 和 2-脱氧-D-葡萄糖导致 LDH 释放逐渐增加，这与时间有关，但与化学浓度无关。1%氧气环境也会随着时间的推移导致细胞损伤，但前提是培养基中不含葡萄糖。与 2-脱氧-D-葡萄糖和暴露于 1%氧气分别在相同的 48 小时时间点引起的 19%和 50%LDH 释放相比，Antimycin A 在诱导氧化应激方面最有效，导致 48 小时时平均 LDH 释放 61%。该细胞培养方法具有以下优点，包括使用无血清培养基和在没有基质支持的情况下生长原代细胞的能力。用于损伤评估的 LDH 测定具有可重复性、成本效益高、客观且最大限度地减少背景细胞死亡。因此，已经建立了一种简单的原代小鼠肾小管上皮细胞培养和损伤方法，为未来的缺血性肾损伤研究提供了有用的工具。
