Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation
Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation
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DOI：
10.1152/ajprenal.00443.2004
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发表时间：
2005-06-01
影响因子：
4.2
通讯作者：
Weinberg, JM
中科院分区：
文献类型：
--
作者：
Feldkamp, T;Kribben, A;Weinberg, JM
在缺氧再氧化（H/R）过程中，近端小管会出现严重的能量缺陷，先前使用荧光电位探针的研究表明，这种缺陷的特征是持续的，部分线粒体失能。为了验证该过程中线粒体失能的主要发生，优化估算系统中线粒体膜电位（δ psi(m)）变化的方法，并阐明缺陷的机制，我们进一步研究了5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑碘化碳菁（JC-1）在这些细胞中的行为，并引入了一种更动态和定量的方法，将safranin O用于小管系统。虽然JC-1的使用可能会因质膜电位降低而变得复杂，从而限制了细胞对JC-1的摄取，而且这种行为在瓦巴因处理的小管中得到了证明，但δ psi(m)的变化完全可以解释红色荧光JC-1聚集体形成的减少以及H/R后观察到的红/绿荧光比率的减少。当小管在摄取JC-1后断电时，红色JC-1聚集体不容易分离，因此不适合进行通电的动态研究。洋地黄苷渗透小管对红花素O的摄取需要非常少的小管，允许在实验操作结束后相对较长时间内测量δ psi(m)，在去电过程中迅速可逆，并允许直接评估底物依赖性、电子传递介导的δ psi(m)和ATP水解支持的δ psi(m)。在H/R后渗透的小管中，用红花素O测量的两种类型的能量都受到了损害，但底物和ATP的结合大大恢复了δ psi(m)。
Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (Delta psi(m)) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in Delta psi(m) entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of Delta psi(m) for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated Delta psi(m), and ATP hydrolysis-supported Delta psi(m). Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored Delta psi(m).