Regulated overexpression of heat shock protein 72 protects Madin-Darby canine kidney cells from the detrimental effects of high urea concentrations

Exposure of renal medullary cells to elevated extracellular NaCl concentrat ions 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 overexpres sed 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 promot er in the eukaryotic expression vector pLK-neo. This construct allowed robu st induction of HSP72 by exposure of stably transfected MDCK cells (MDCK-LK 72) to 0.1 muM DEX. Increased HSP72 abundance significantly improved surviv al 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 signifi cant effect on HSP72 abundance or urea resistance. In accordance with those findings, lactate dehydrogenase activity in the supernatant was significan tly reduced, compared with appropriate control samples, only in MDCK-LK72 c ells overexpressing HSP72. Labeling with annexin V-FITC and propidium iodid e, followed by flow cytometry, revealed that overexpression of HSP72 was as sociated with a reduction in the number of apoptotic-lysed cells, a concomi tant retardation of apoptosis, and an increase in the number of viable cell s. These data support the view that HSP72, which is very abundant in the re nal inner medulla, is an important component of the defense mechanism of me dullary cells against extreme concentrations of urea.