Inhibition of NaCl-induced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations

Exposure of Madin-Darby canine kidney (MDCK) cells to elevated extracellula r NaCl concentrations is associated with increased heat shock protein 72 (H SP72) expression and improved survival of these pretreated cells upon expos ure to an additional 600 mM urea in the medium. To establish a causal relat ionship between HSP72 expression and cell protection against high urea conc entrations, two approaches to inhibit NaCl-induced HSP72 synthesis prior to exposure to 600 mM urea were employed. First, the highly specific p38 kina se inhibitor SB203580 was added (100 mu M) to the hypertonic medium (600 mo sm/kg H2O by NaCl addition, 2 days of exposure), which significantly reduce d HSP72 mRNA abundance and HSP72 content. Survival of these cells after a 2 4-h urea treatment (600 mM) was markedly curtailed compared with appropriat e controls. Second, a pcDNA3-based construct, containing 322 bases of the H SP72 open reading frame in antisense orientation and the geneticine resista nce gene, was transfected into MDCK cells. Clones with strong inhibition of HSP72 synthesis and others which express the protein at normal levels (com parable to nontransfected MDCK cells) after heat shock treatment or hyperto nic stress were established. When these transformants were subjected to hyp ertonic stress for 2 days prior to exposure to an additional 600 mM urea fo r 24 h, cell survival was significantly reduced in those clones in which HS P72 expression was strongly inhibited. These results provide further eviden ce for the protective function of HSP72 against high urea concentrations in renal epithelial cells.