Abstract 1950 Nephrology Platform, Sunday, 5/2

Ischemic renal injury consists of at least two major stresses: i) a decrease of oxygen (O2) supply (hypoxia) and ii) limited availability of cellular energy (ATP depletion). Each of these injuries is believed to rapidly activate stress-sensing transcription factors. Therefore, in LLC-PK1 cells in vitro and ischemic rat kidneys in vivo, we investigated activation of two transcription factors: HSF which is known to induce production of a variety of heat-shock proteins and HIF which orchestrates and modulates the molecular response to hypoxia. Cells were grown to confluence, serum deprived for 24 hours, and then subjected to either hypoxia or substrate depletion. Hypoxia was achieved by incubation in 0.1% - 5% O2. Substrate depletion occurred when Antimycin-A (a mitochondrial inhibitor) was added to serum and glucose-free media. HSF and HIF activation were analyzed by gel shift assays using nuclear extracts incubated with radiolabeled oligonucleotides comprising the HSE (heat shock element) or HRE (hypoxia-response element), respectively.

In hypoxic cells, increased HIF binding activity was observed as early as 2 hours and persisted for 24 hours, but no HSF activation was detected during or after 24 hours of hypoxia. In contrast, rapid activation of HSF was detected as early as 30 minutes after substrate depletion, but no HIF activation was detected even after 8 hours of substrate depletion. In rats, 45 minutes of renal ischemia, in vivo, was found to activate both HIF and HSF.

Thus, this study suggests that: 1) in renal cells, HIF acts as a stress-sensing transcription factor activated by changes in O2 but not metabolic alteration, whereas, 2) HSF primarily responds to metabolic stress associated with ATP depletion but not hypoxia. Moreover, renal ischemia in vivo activates both of these biologically important stress-response pathways, suggesting that injury and recovery from ischemia in vivo may be more complex than reflected in renal cells in vitro.