Nephrology Dialysis Transplantation, Vol 12, Issue 12 2549-2556, Copyright © 1997 by Oxford University Press
M Tijsen, S Peters, R Bindels, C van Os and J Wetzels
Background: Isolated rat proximal tubules are
frequently used as a model to study hypoxic injury. Glycine is a very
effective protective agent against hypoxia-induced cell injury in this
model. The mechanisms involved in hypoxic renal injury and glycine
protection are still debated. We have focused on the role of proteolytic
enzymes. Methods: Isolated rat proximal tubules in
suspension were gassed with either 95%O2/5%CO2 or 95%N2/5%CO2 to create
normoxic or hypoxic conditions. Cell injury was assessed by the release of
LDH. Activity of proteolytic enzymes was measured by quantifying the
release of fluorescent 7-amino-4-methylcoumarin from specific substrates,
which were added to tubules in suspension or to cytosolic fractions of
permeabilized tubules. Results: Fifteen minutes of
hypoxia caused cell injury, which was completely prevented by glycine.
Activities of serine-, aspartate-, and the calcium-dependent cysteine
protease calpain were increased in these hypoxic tubules in suspension, but
only calpain activity was attenuated by glycine. Cytosolic fractions
obtained by digitonin-permeablization of hypoxic (15 min) tubules showed
increased proteolytic activity of all measured classes of proteases and
glycine prevented these increases. In measurements performed at an earlier
time point (7.5 min) neither changes in calpain activity nor effects of
glycine were detected. Calpain activity was not inhibited directly by
glycine. Conclusions: Hypoxia increases the activity
of several classes of proteases. The effects of glycine on protease
activation are equivocal, and may merely reflect the potential of glycine
to prevent hypoxia-induced lethal membrane injury. Key
words: calpain; cell injury; glycine; hypoxia; protease
ORIGINAL ARTICLES
Glycine protection against hypoxic injury in isolated rat proximal tubules: the role of proteases
Department of Medicine, Division of Nephrology and Department of Cell Physiology, University Hospital Nijmegen, Nijmegen, The Netherlands; Corresponding author at: 545 Department of Nephrology, University Hospital Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands
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