Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments.
Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments.
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DOI：
10.1152/ajprenal.00031.2020
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发表时间：
2020-03
期刊：
影响因子：
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通讯作者：
Allison N. Mccrimmon;Mark Domondon;Regina F. Sultanova;D. Ilatovskaya;K. Stadler
中科院分区：
文献类型：
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作者：
Allison N. Mccrimmon;Mark Domondon;Regina F. Sultanova;D. Ilatovskaya;K. Stadler
线粒体功能的改变是许多肾脏疾病的核心，包括急性损伤、糖尿病肾病、高血压和慢性肾脏疾病。因此，越来越需要可靠和快速的方法来评估肾细胞线粒体的呼吸功能。然而，尽管培养细胞或分离的线粒体对于许多机械研究来说是不可或缺的，但它们在体内或接近体内的情况下往往不能重现。与体内或体外的情况相比，培养的和/或永生化的细胞经常改变其生物能量特征和表型，分离的线粒体被简单地从细胞环境中移除。这对于极其复杂的器官，如肾脏，尤其重要。在这里，我们报告了一种使用安捷伦海马XFe24和XF96细胞外通量分析仪快速评估新鲜分离的肾小球或近端小管(PT)片段线粒体氧耗的新方法的开发和验证。我们在几个健康和患病的啮齿动物模型上验证了这项技术-C57BL/6J小鼠、糖尿病db/db小鼠及其匹配的db/+对照组和Dahl盐敏感大鼠。我们将这些数据与分离的线粒体的呼吸进行了比较。该方法可用于研究人员选择的任何啮齿动物模型的线粒体耗氧量的快速评估。本文介绍的分离方法确保了活性和功能性的PT片段和肾小球，具有保存的细胞环境，以便在其周围环境和相互作用的背景下研究线粒体的功能。
Changes in mitochondrial function are central to many forms of kidney disease including acute injury, diabetic nephropathy, hypertension and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close-to-in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared to in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here we report the development and validation of a new approach for rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular (PT) fragments using the Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models - the C57BL/6J mouse, the diabetic db/db mouse and their matching db/+ control and the Dahl salt sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator's choice. The isolation methods presented here ensure viable and functional PT fragments and glomeruli, with preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.