Wiegele G, Brandis M, Zimmerhackl L B
Department of Pediatrics, Albert-Ludwigs-University, Freiburg, Germany.
Nephrol Dial Transplant. 1998 May;13(5):1158-67. doi: 10.1093/ndt/13.5.1158.
Ischaemia is the most frequent cause of acute renal failure. It has been previously demonstrated that ischaemia is connected with signs of necrosis and apoptosis. Apoptosis is an energy-dependent process. During ischaemia intercellular energy levels decline rapidly. Therefore, the goal of the investigation was to reveal the time dependency of cell death mechanisms during ischaemia leading to irreversibility of tissue damage.
A model of renal ischaemia induced by ATP depletion was used in LLC-PK1 and MDCK-cells. Cell proliferation, determined by 3H-thymidine and BrdU incorporation and by the Ki67-labelling index was affected already after 1-2 h of ATP depletion in both cell lines. Cell viability and membrane leakage, estimated by trypan blue and propidium iodide exclusion and LDH release, was profoundly increased after 8-16 h. Evaluation of mechanisms of necrotic or apoptotic cell death was calculated from fraction of cells with pyknotic nuclei, investigation of DNA fragmentation and, by translocation of phosphatidylserine (PS) from the inner membrane face to the surface. In both cell lines increased numbers of cells with condensed nuclei was not a major sign of apoptosis. Only in MDCK cells were the numbers of cells with condensed nuclei significantly increased after 1 h compared to controls. As a hallmark of apoptosis, ATP depletion resulted in intranucleosomal DNA fragmentation after 1 h. After 8-16 h this pattern changed to a smear pattern, as a sign for necrosis. PS staining was detectable at the cell surface after 1 h.
Ischaemia is associated with a rapid loss of proliferation and signs of apoptosis at early stages in a small proportion of cells. Most cells undergo the necrotic pathway of cell death after prolonged ATP depletion (8 h). There was no difference in behaviour comparing proximal (LLC-PK1) with more distal (MDCK) cell culture models. These results may help to explain the findings that apoptosis and necrosis have both been described after renal ischaemia.
缺血是急性肾衰竭最常见的病因。先前已证明，缺血与坏死和凋亡迹象相关。凋亡是一个能量依赖过程。在缺血期间，细胞间能量水平迅速下降。因此，本研究的目的是揭示缺血期间导致组织损伤不可逆的细胞死亡机制的时间依赖性。
在LLC-PK1和MDCK细胞中使用ATP耗竭诱导的肾缺血模型。通过3H-胸腺嘧啶核苷和BrdU掺入以及Ki67标记指数测定的细胞增殖在两种细胞系中ATP耗竭1-2小时后就已受到影响。通过台盼蓝和碘化丙啶排斥以及乳酸脱氢酶释放估计的细胞活力和膜通透性在8-16小时后显著增加。通过对核固缩细胞比例的计算、DNA片段化研究以及磷脂酰丝氨酸（PS）从内膜面向表面的易位来评估坏死或凋亡性细胞死亡的机制。在两种细胞系中，核固缩细胞数量增加并非凋亡的主要标志。仅在MDCK细胞中，与对照相比，1小时后核固缩细胞数量显著增加。作为凋亡的标志，ATP耗竭1小时后导致核小体间DNA片段化。8-16小时后，这种模式转变为拖尾模式，这是坏死的标志。1小时后在细胞表面可检测到PS染色。
缺血与早期少数细胞增殖迅速丧失和凋亡迹象相关。大多数细胞在长时间ATP耗竭（8小时）后经历坏死性细胞死亡途径。比较近端（LLC-PK1）和远端（MDCK）细胞培养模型，其行为没有差异。这些结果可能有助于解释肾缺血后既有凋亡又有坏死的发现。
