Iwata M, Myerson D, Torok-Storb B, Zager R A
Department of Medicine, University of Washington, Seattle.
J Am Soc Nephrol. 1994 Dec;5(6):1307-13. doi: 10.1681/ASN.V561307.
It has recently been suggested that endonuclease activation and/or apoptosis, possibly triggered by oxidant stress, are important pathogenetic mechanisms in oxygen deprivation/reoxygenation-induced proximal tubular cell death. To explore this possibility, DNA "laddering," a characteristic feature of these processes, was sought in: (1) postischemic rat kidneys (25- or 40-min arterial clamping; 0, 1, 4, 8, 24, and 48 h and 6 days reflow); (2) posthypoxic isolate rat proximal tubular segments and (3) cultured human kidney proximal tubular cells (HK-2) subjected either to energy depletion plus Ca2+ overload (antimycin A plus 2-deoxyglucose plus Ca2+ ionophore A23187), or to H2O2-induced cell death. DNA was subsequently extracted, electrophoresed through agarose gels, and visualized with ethidium bromide or Southern blotting. To maximize ladder detection, DNA samples were also end-labeled with 32P dideoxyadenosine triphosphate with terminal deoxynucleotidyl transferase (tdt), followed by electrophoresis. None of the postischemic DNA samples demonstrated any laddering by either ethidium bromide staining or Southern analysis (apoptotic lymphocyte DNA was a positive control). However, trace laddering was apparent by the tat technique, commencing at 1 h of reflow, peaking at 24 h, and resolving slowly thereafter. This finding correlated with the morphologic expression of tubular necrosis, not apoptosis. Hypoxia/reoxygenation caused proximal tubular segment death (44 to 64%), and HK-2 cells were slowly killed by both the H2O2 and the energy depletion/Ca(2+)-loading protocols. However, neither protocol induced ethidium bromide- or tdt-detectable DNA laddering. It was concluded that: (1) minimal DNA laddering develops postischemia, and this change is reliably detected only by the tdt method; (2) it correlates with the morphologic expression of tubular necrosis, not apoptosis; and (3) in vitro oxidative- and energy depletion-mediated proximal tubular cell death can be dissociated from DNA ladder formation.
最近有人提出，内切核酸酶激活和/或凋亡可能由氧化应激触发，是缺氧/复氧诱导近端肾小管细胞死亡的重要发病机制。为了探究这种可能性，研究人员在以下实验中寻找DNA“梯形条带”（这些过程的一个特征）：（1）缺血后大鼠肾脏（动脉夹闭25或40分钟；再灌注0、1、4、8、24、48小时和6天）；（2）缺氧后分离的大鼠近端肾小管节段；（3）培养的人肾近端肾小管细胞（HK - 2），分别使其经历能量耗竭加Ca2+过载（抗霉素A加2 - 脱氧葡萄糖加Ca2+离子载体A23187）或H2O2诱导的细胞死亡。随后提取DNA，通过琼脂糖凝胶电泳，并用溴化乙锭或Southern印迹法进行可视化分析。为了最大限度地检测到梯形条带，DNA样品还用32P双脱氧三磷酸腺苷与末端脱氧核苷酸转移酶（tdt）进行末端标记，然后进行电泳。无论是通过溴化乙锭染色还是Southern分析，缺血后DNA样品均未显示任何梯形条带（凋亡淋巴细胞DNA作为阳性对照）。然而，通过tdt技术在再灌注1小时时可明显观察到微量梯形条带，在24小时达到峰值，此后逐渐消失。这一发现与肾小管坏死而非凋亡的形态学表现相关。缺氧/复氧导致近端肾小管节段死亡（44%至64%），H2O2和能量耗竭/Ca(2+)加载方案均缓慢杀死HK - 2细胞。然而，两种方案均未诱导溴化乙锭或tdt可检测到的DNA梯形条带。研究得出以下结论：（1）缺血后出现最小程度的DNA梯形条带，且这种变化仅通过tdt方法才能可靠检测到；（2）它与肾小管坏死而非凋亡的形态学表现相关；（3）体外氧化和能量耗竭介导的近端肾小管细胞死亡与DNA梯形条带形成无关。
