Nephrology Dialysis Transplantation, Vol 13, Issue 5 1158-1167, Copyright © 1998 by Oxford University Press
G Wiegele, M Brandis and L Zimmerhackl
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
K167-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. Key words: ATP depletion; cell
proliferation; acute renal failure; cell death
ORIGINAL ARTICLES
Apoptosis and necrosis during ischaemia in renal tubular cells (LLC-PK1 and MDCK)
Department of Pediatrics, Albert-Ludwigs-University, Freiburg, Germany; Corresponding author at: Univ.-Kinderklinik, Mathildenstrasse 1, D-79106 Freiburg, Germany
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