Rump Katharina, Unterberg Matthias, Bergmann Lars, Bankfalvi Agnes, Menon Anil, Schäfer Simon, Scherag André, Bazzi Zainab, Siffert Winfried, Peters Jürgen, Adamzik Michael
Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer, In der Schornau 55, 45882, Bochum, Germany.
Institut für Pharmakogenetik, Universität Duisburg-Essen, Duisburg, Germany.
J Transl Med. 2016 Nov 21;14(1):321. doi: 10.1186/s12967-016-1079-2.
The C-allele of the aquaporin (AQP5) -1364A/C polymorphism is associated with decreased AQP5 expression but increased 30-day survival in patients with severe sepsis. AQP5 expression might affect survival via an impact on cell migration. Consequently, we tested the hypothesis that (1) Aqp5 knockout (KO) compared to wild type (WT) mice show an increased survival following lipopolysaccharide (LPS) administration, and that (2) AQP5 expression and the AQP5 -1364A/C polymorphism alters immune cell migration.
We investigated Aqp5-KO and wild type mice after intraperitoneal injection of either E.coli lipopolysaccharide (LPS, serotype O127:B8, 20 mg/kg) or saline. Furthermore, neutrophils of volunteers with the AA-AQP5 or AC/CC-AQP5- genotype were incubated with 10 M Chemotactic peptide (fMLP) and their migration was assessed by a filter migration assay. Additionally, AQP5 expression after fMLP incubation was analyzed by RT-PCR and Western blot. Moreover, migration of AQP5 overexpressing Jurkat cells was studied after SDF-1α-stimulation. We used exact Wilcoxon-Mann-Whitney tests; exact Wilcoxon signed-rank tests and the Kaplan-Meier estimator for statistical analysis.
Fifty-six percent of Aqp5-KO but only 22% of WT mice survived following LPS-injection. WT mice showed increased neutrophil migration into peritoneum and lung compared to Aqp5-KO mice. Target-oriented migration of neutrophils was seen after 0.5 h in AA-genotype cells but only after 1.5 h in AC/CC-genotype cells, with a threefold lower migrating cell count. AQP5 overexpressing Jurkat cells showed a 2.4 times stronger migration compared to native Jurkat cells.
The AQP5 genotype may influence survival following LPS by altering neutrophil cell migration. Trial registration DRKS00010437. Retrospectively registered 26 April 2016.
水通道蛋白（AQP5）-1364A/C多态性的C等位基因与严重脓毒症患者AQP5表达降低但30天生存率增加相关。AQP5表达可能通过影响细胞迁移来影响生存率。因此，我们检验了以下假设：（1）与野生型（WT）小鼠相比，水通道蛋白5基因敲除（KO）小鼠在给予脂多糖（LPS）后生存率增加；（2）AQP5表达及AQP5 -1364A/C多态性改变免疫细胞迁移。
我们在腹腔注射大肠杆菌脂多糖（LPS，血清型O127:B8，20mg/kg）或生理盐水后研究了AQP5基因敲除小鼠和野生型小鼠。此外，将AA-AQP5或AC/CC-AQP5基因型志愿者的中性粒细胞与10μM趋化肽（fMLP）孵育，并通过滤膜迁移试验评估其迁移情况。另外，通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法分析fMLP孵育后的AQP5表达。此外，在基质细胞衍生因子-1α（SDF-1α）刺激后研究了过表达AQP5的人急性T淋巴细胞白血病细胞（Jurkat细胞）的迁移。我们使用精确的Wilcoxon-Mann-Whitney检验、精确的Wilcoxon符号秩检验和Kaplan-Meier估计量进行统计分析。
LPS注射后，56%的AQP5基因敲除小鼠存活，但野生型小鼠仅22%存活。与AQP5基因敲除小鼠相比，野生型小鼠中性粒细胞向腹膜和肺的迁移增加。AA基因型细胞在0.5小时后可见中性粒细胞的定向迁移，但AC/CC基因型细胞仅在1.5小时后可见，迁移细胞计数低三倍。与天然Jurkat细胞相比，过表达AQP5的Jurkat细胞迁移能力强2.4倍。
AQP5基因型可能通过改变中性粒细胞迁移来影响LPS刺激后的生存率。试验注册号DRKS00010437。于2016年4月26日进行回顾性注册。
