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Apoptosis and necrosis during ischaemia in renal tubular cells (LLC-PK1 and MDCK).

BACKGROUND
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.


METHODS AND RESULTS
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.


CONCLUSIONS
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.