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Department of Physiology, University of Munich, Munich, Germany.
Correspondence to Dr. Wolfgang Neuhofer, Department of Physiology, University of Munich, Pettenkoferstrasse 12, 80336 München, Germany. Phone: +49-89-5996-534; Fax: +49-89-5996-532; E-mail: W.Neuhofer{at}physiol.med.uni-muenchen.de
ABSTRACT. Exposure of renal medullary cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved resistance to subsequent exposure to a high urea concentration (600 mM). To establish a causal relationship between HSP72 expression and protection against high urea concentrations, HSP72 was inducibly overexpressed in Madin-Darby canine kidney (MDCK) cells, in the absence of hypertonic stress before urea exposure. For this purpose, the human stress-inducible HSP72 gene was cloned downstream from a dexamethasone (DEX)-inducible promoter in the eukaryotic expression vector pLKneo. This construct allowed robust induction of HSP72 by exposure of stably transfected MDCK cells (MDCK-LK72) to 0.1 µM DEX. Increased HSP72 abundance significantly improved survival rates after 24-h exposure of the cells to medium containing 600 mM urea (14 versus 43%). In mock-transfected or wild-type cells, DEX had no significant effect on HSP72 abundance or urea resistance. In accordance with those findings, lactate dehydrogenase activity in the supernatant was significantly reduced, compared with appropriate control samples, only in MDCK-LK72 cells overexpressing HSP72. Labeling with annexin V-FITC and propidium iodide, followed by flow cytometry, revealed that overexpression of HSP72 was associated with a reduction in the number of apoptotic-lysed cells, a concomitant retardation of apoptosis, and an increase in the number of viable cells. These data support the view that HSP72, which is very abundant in the renal inner medulla, is an important component of the defense mechanism of medullary cells against extreme concentrations of urea.
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