HYPOTONICITY INCREASES TRANSCRIPTION, EXPRESSION, AND ACTION OF EGR-1IN MURINE RENAL MEDULLARY MIMCD3 CELLS

In cells of the murine renal inner medullary collecting duct (mIMCD3) cell line, acute hypotonic shock (50% dilution of medium with sterile water but not with sterile 150 mM NaCl) increased Egr-1 mRNA abundance 2.5-fold at 6 h, as determined by Northern analysis. This increase wa s accompanied by increased Egr-1 transcription, as quantitated by luci ferase reporter gene assay. Increased transcription was dose dependent , additive with other Egr-1 transcriptional activators, and occurred i n the absence of overt cytotoxicy, as quantitated via a fluorometric v iability assay. In addition, hypotonic stress increased Egr-1 protein abundance, which was accompanied by augmented Egr-1-specific DNA bindi ng ability, as measured via electrophoretic mobility shift assay. Incr eased DNA binding was further associated with increased transactivatio n by Egr-1, demonstrated through transient transfection of mIMCD3 cell s with a luciferase reporter gene driven by tandem repeats of the Egr- 1 DNA consensus sequence. Taken together, these data indicate that hyp otonic stress activates Egr-1 transcription, translation, DNA binding, and transactivation in renal medullary cells. This phenomenon might p lay a role in the acquisition of the adaptive phenotype in response to hypotonic stress in cells of the renal medulla in vivo.