K+-induced HSP-72 expression is mediated via rapid Ca2+ influx in renal epithelial cells

Abstract

Pathophysiological stimuli, including hypoxia, lead to K+ efflux from the intracellular lumen to the extracellular space, thereby increasing local tissue K+ concentrations and depolarizing resident cells. In this study, we investigated the effects of increased extracellular K+ concentrations ([K+]e) on heat shock protein (HSP) expression in the porcine proximal tubule epithelial cell line LLC-PK1. We analyzed HSP-25, HSP-72, HSC-73, and HSP-90 protein expression by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene assays using the proximal 1,440 bp of the HSP-72 promoter. Elevating [K+]e from 20 to 50 mM increased HSP-72 protein expression and promoter activity but did not affect HSP-25, HSC-73, or HSP-90 levels. Addition of identical concentrations of sodium chloride did not increase HSP-72 expression to a similar amount. The Ca2+ channel blocker diltiazem and the Ca2+-specific chelator EGTA-AM abolished high [K+]e-induced HSP-72 expression by 69.7 and 75.2%, respectively, indicating that the transcriptional induction of HSP-72 involves Ca2+ influx. As measured by confocal microscopy using the Ca2+ dye fluo 3-AM, we also observed a rapid increase of intracellular Ca2+ concentration as early as 30 s after placing LLC-PK1 cells in high [K+]e. We further analyzed whether Ca2+ influx was necessary for induction of HSP-72 expression by high [K+]e using Ca2+-free medium. Here, induction of HSP-72 in response to high [K+]e was completely abolished. Our data thus demonstrate activation of a protective cellular response to ionic stress, e.g., elevated K+ concentrations, by specifically increasing protein levels of HSP-72.