DNA methylation of a NF-\(\kappa\)B binding site in the aquaporin 5 promoter impacts on mortality in sepsis

Altered aquaporin 5 (\(\it AQP5\)) expression in immune cells impacts on key mechanisms of inflammation and is associated with sepsis survival. Since epigenetic regulation via DNA methylation might contribute to a differential \(\it AQP5\) expression in sepsis, we tested the hypotheses that DNA methylation of the \(\it AQP5\) promotor (1) influences \(\it AQP5\) expression, (2) is associated with the 30-day survival of septic patients, and (3) alters the nuclear transcription factor NF-\(\kappa\)B binding. \(\it AQP5\) mRNA expression was quantified by real-time PCR in whole blood samples of 135 septic patients. \(\textit {In silico}\) computer analysis of the \(\it AQP5\) promoter (nt-567 to nt-975) revealed seven putative inflammatory transcription factor binding sites and methylation of these sites was analyzed. Electrophoretic mobility shift assays were performed to assess the binding of nuclear NF-\(\kappa\)B to the \(\it AQP5\) promoter region nt-937. After adjustment for multiple testing, a greater methylation rate was found at cytosine site nt-937 in the \(\it AQP5\) promoter linked to NF-\(\kappa\)B binding in non-survivors compared to survivors (p = 0.002, p\(_{adj}\) = 0.014). This was associated with greater \(\it AQP5\) mRNA expression in non-survivors (p = 0.037). Greater (\(\geq\)16%) promoter methylation at nt-937 was also associated with an independently increased risk of death within 30 days (HR: 3.31; 95% CI: 1.54–6.23; p = 0.002). We detected a functionally important \(\it AQP5\) promoter cytosine site (nt-937) linked to the binding of the inflammatorily acting nuclear transcription factor NF-\(\kappa\)B, with increased methylation in sepsis non-survivors. Thus, nt-937 \(\it AQP5\) promoter methylation, presumably related to NF-\(\kappa\)B binding, is prognostically relevant in sepsis and demonstrates that epigenetic changes impact on sepsis outcome.