DNA methylation of a NF-\u03baB binding site in the aquaporin 5 promoter impacts on mortality in sepsis

Katharina Rump,Jürgen Peters,Michael Adamzik,Tim Rahmel,Björn Koos,Matthias Unterberg,Agnes Dahlke,Lars Bergmann,Hartmuth Nowak,Simon T Schäfer,Winfried Siffert

doi:10.1038/s41598-019-55051-8

Katharina Rump, Jürgen Peters + Show 9 more

Open Access

https://doi.org/10.1038/s41598-019-55051-8

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Abstract

Altered aquaporin 5 (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 AQP5 expression in sepsis, we tested the hypotheses that DNA methylation of the AQP5 promotor (1) influences AQP5 expression, (2) is associated with the 30-day survival of septic patients, and (3) alters the nuclear transcription factor NF-κB binding. AQP5 mRNA expression was quantified by real-time PCR in whole blood samples of 135 septic patients. In silico computer analysis of the 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-κB to the AQP5 promoter region nt-937. After adjustment for multiple testing, a greater methylation rate was found at cytosine site nt-937 in the AQP5 promoter linked to NF-κB binding in non-survivors compared to survivors (p = 0.002, padj = 0.014). This was associated with greater AQP5 mRNA expression in non-survivors (p = 0.037). Greater (≥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 AQP5 promoter cytosine site (nt-937) linked to the binding of the inflammatorily acting nuclear transcription factor NF-κB, with increased methylation in sepsis non-survivors. Thus, nt-937 APQ5 promoter methylation, presumably related to NF-κB binding, is prognostically relevant in sepsis and demonstrates that epigenetic changes impact on sepsis outcome.

Highlights

Sepsis is a grave medical condition and its mortality remains high[1]
aquaporin 5 (AQP5) is involved in a lot of pathophysiological properties that prevail in sepsis and its altered expression seems to represent a crucial regulatory mechanism[2]
No evidence for significant associations of 30-day survival was found for age (p = 0.757), sex (p = 0.855), body mass index (p = 0.128), necessity for continuous hemofiltration/dialysis (p = 0.129) and Simplified Acute Physiology Score II (p = 0.119)

Summary

Introduction

Sepsis is a grave medical condition and its mortality remains high[1]. identification of diagnostic and therapeutic targets is a corner-stone of current research. The AA-genotypes of the AQP5 -1364A/C associated with greater AQP5 expression showed increased pulmonary inflammation and an increased risk of acute kidney injury in ARDS13,15 These results suggest a protective impact of lesser AQP5 expression in proinflammatory diseases, and mechanisms linked to an altered AQP5 expression are of great interest. In the context of sepsis, there is growing evidence that epigenetic modifications can affect protein expression[19,20,21]; AQP5 promoter methylation might be a mechanism influencing AQP5 expression. It is unknown whether AQP5 expression under septic conditions might be epigenetically regulated by an altered promoter methylation. We tested the hypotheses that DNA methylation at a specific AQP5 promoter binding site is associated (1) with altered AQP5 expression, (2) 30-day survival of septic patients and (3) altered NF-κB binding

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