Major adverse kidney events are associated with the aquaporin 5-1364A/C promoter polymorphism in sepsis

Since the functionally important $\it AQP5$ -1364A/C single nucleotide promoter polymorphism alters key mechanisms of inflammation and survival in sepsis, it may affect the risk of an acute kidney injury. Accordingly, we tested the hypothesis in septic patients that this $\it AQP5$ polymorphism is associated with major adverse kidney events and also validated its impact on 90-day survival. In this prospective observational monocentric genetic association study 282 septic patients were included and genotyped for the $\it AQP5$ –1364A/C polymorphism (rs3759129). The primary endpoint was the development of major adverse kidney events within 30 days. In AC/CC genotypes, major adverse kidney events were less frequent (41.7%) than in AA genotypes (74.3%; OR:0.34; 95%-CI: 0.18–0.62; $\it p$ < 0.001). Ninety-day survival was also associated with the $\it AQP5$ polymorphism ($\it p$ = 0.004), with 94/167 deaths (56.3%) in AA genotypes, but only 46/115 deaths (40.0%) in C-allele carriers. Multiple proportional hazard analysis revealed AC/CC genotypes to be at significantly lower risk for death within 90 days (HR: 0.60; 95%-CI: 0.42-0.86; $\it p$ = 0.006). These findings confirm the important role of the AQP5 -1364A/C polymorphism as an independent prognostic factor in sepsis. Furthermore, we demonstrate a strong association between this $\it AQP5$ polymorphism and susceptibility for major adverse kidney events suggesting a promising characteristic in terms of precision medicine

Phrenic nerve block caused by interscalene brachial plexus block

$\bf Background:$ Interscalene brachial plexus (ISB) block is often associated with phrenic nerve block and diaphragmatic paresis. The goal of our study was to test if the anterior or the posterior ultrasound guided approach of the ISB is associated with a lower incidence of phrenic nerve blocks and impaired lung function. $\bf Methods:$ This was a prospective, randomized and single-blinded study of 84 patients scheduled for elective shoulder surgery who fullfilled the inclusion and exclusion critereria. Patients were randomized in two groups to receive either the anterior ($\it n$ = 42) or the posterior ($\it n$ = 42) approach for ISB. Clinical data were recorded. In both groups patients received ISB with a total injection volume of 15 ml of ropivacaine 1 %. Spirometry was conducted at baseline (T$_{0}$) and 30 min (T$_{30}$) after accomplishing the block. Changes in spirometrical variables between T$_{0}$ and T$_{30}$ were investigated by Wilcoxon signed-rank test for each puncture approach. The temporal difference between the posterior and the anterior puncture approach groups were again analyzed by the Wilcoxon-Mann-Whitney test. $\bf Results:$ The spirometric results showed a significant decrease in vital capacity, forced expiratory volume per second, and maximum nasal inspiratory breathing after the Interscalene brachial plexus block; indicating a phrenic nerve block ($\it p$ <0.001, Wilcoxon signed-rank). A significant difference in the development of the spirometric parameters between the anterior and the posterior group could not be identified (Wilcoxon-Mann-Whitney test). Despite the changes in spirometry, no cases of dyspnea were reported. $\bf Conclusion:$ A different site of injection (anterior or posterior) did not show an effect in reducing the cervical block spread of the local anesthetic and the incidence of phrenic nerve blocks during during ultrasound guided Interscalene brachial plexus block. Clinical breathing effects of phrenic nerve blocks are, however, usually well compensated, and subjective dyspnea did not occur in our patients

The aquaporin 5 −1364A/C promoter polymorphism is associated with cytomegalovirus infection risk in kidney transplant recipients

$\bf Background:$ The aquaporin 5 (AQP5) −1364A/C promoter single nucleotide polymorphism affects key mechanisms of inflammation and immune cell migration. Thus, it could be involved in the pathogenesis of cytomegalovirus infection. Accordingly, we tested the hypothesis that the AQP5 promoter −1364A/C polymorphism is associated with the risk of cytomegalovirus infection in kidney transplantation recipients. $\bf Methods:$ We included 259 adult patients who received a kidney transplant from 2007 and 2014 in this observational study. Patients were genotyped for the AQP5 promoter −1364A/C single nucleotide polymorphism and followed up for 12 months after transplantation. Kaplan–Meier plots and multivariable proportional hazard analyses were used to evaluate the relationship between genotypes and the incidence of cytomegalovirus infection. $\bf Results:$ The incidences of cytomegalovirus infection within 12 months after kidney transplantation were 22.9% for the AA genotypes (43/188) and 42.3% for the AC/CC genotypes (30/71; p = 0.002). Furthermore, multivariable COX regression revealed the C-allele of the AQP5 −1364A/C polymorphism to be a strong and independent risk factor for cytomegalovirus infection. In this analysis, AC/CC subjects demonstrated a more than 2-fold increased risk for cytomegalovirus infection within the first year after kidney transplantation (hazard ratio: 2.28; 95% CI: 1.40–3.73; p = 0.001) compared to that in individuals with homozygous AA genotypes. $\bf Conclusions:$ With respect to opportunistic cytomegalovirus infections (attributable to immunosuppression after kidney transplantation), the C-allele of the AQP5 −1364A/C promoter polymorphism is independently associated with an increased 12-months infection risk. These findings emphasize the importance of genetic variations as additional risk factors of cytomegalovirus infection after solid organ transplantations and might also facilitate the discovery of novel therapeutic targets

The aquaporin 3 promoter polymorphism − 1431 A/G is associated with acute graft rejection and cytomegalovirus infection in kidney recipients due to altered immune cell migration

Major complications after kidney transplantation are graft rejection and cytomegalovirus (CMV) infection, which are related to T-cell function, which depends on aquaporin 3 (AQP3) expression. The impact of the AQP3 A(−1431)G promoter polymorphism in kidney transplant recipients was unelucidated and we explored the effect of AQP3 polymorphism on immune cell function and its association with graft rejection and CMV infection in 237 adult patients within 12 months after transplantation. AQP3 promoter polymorphism was molecular and functional characterized. Kaplan–Meier plots evaluated the relationship between genotypes and the incidence of CMV infection and graft rejection. AQP3 A(−1431)G A-allele was associated with enhanced immune cell migration and AQP3 expression in T-cells. The incidences of rejection were 45.4% for the A-allele and 27.1% for G-allele carriers ($\it p$ = 0.005) and the A-allele was a strong risk factor (hazard ratio (HR): 1.95; 95% CI: 1.216 to 3.127; $\it p$ = 0.006). The incidences for CMV infection were 21% for A-allele and 35% for G-allele carriers ($\it p$ = 0.013) and G-allele was an independent risk factor ($\it p$ = 0.023), with a doubled risk for CMV infection (HR: 1.9; 95% CI: 1.154 to 3.128; $\it p$ = 0.012). Hence, A-allele confers more resistance against CMV infection, but susceptibility to graft rejection mediated by T-cells. Thus, AQP3-genotype adapted management of immunosuppression and antiviral prophylaxis after kidney transplantation seems prudent

Long-term mortality and outcome in hospital survivors of septic shock, sepsis, and severe infections

Patients with severe infections and especially sepsis have a high in-hospital mortality, but even hospital survivors face long-term sequelae, decreased health-related quality of life, and high risk of death, suggesting a great need for specialized aftercare. However, data regarding a potential benefit of post-discharge rehabilitation in these patients are scarce. In this retrospective matched cohort study the claim data of a large German statutory health care insurer was analyzed. 83,974 hospital survivors having suffered from septic shock, sepsis, and severe infections within the years 2009–2016 were identified using an ICD abstraction strategy closely matched to the current Sepsis-3 definition. Cases were analyzed and compared with their matched pairs to determine their 5-year mortality and the impact of post-discharge rehabilitation. Five years after hospital discharge, mortality of initial hospital survivors were still increased after septic shock ($HR_{adj}$ 2.03, 95%-CI 1.87 to 2.19; $\it P$<0.001), sepsis ($HR_{adj}$ 1.73, 95%-CI 1.71 to 1.76; $\it P$<0.001), and also in survivors of severe infections without organ dysfunction ($HR_{adj}$ 1.70, 95%-CI 1.65 to 1.74; $\it P$<0.001) compared to matched controls without infectious diseases. Strikingly, patients treated in rehabilitation facilities showed a significantly improved 5-year survival after suffering from sepsis or septic shock ($HR_{adj}$ 0.81, 95%-CI 0.77 to 0.85; $\it P$<0.001) as well as severe infections without organ dysfunction ($HR_{adj}$ 0.81, 95%-CI 0.73 to 0.90; $\it P$<0.001) compared to matched patients discharged to home or self-care. Long-term mortality and morbidity of hospital survivors are markedly increased after septic shock, sepsis and severe infections without organ dysfunction, but best 5-year survival was recorded in patients discharged to a rehabilitation facility in all three groups. Thus, our data suggest that specialized aftercare programs may help to improve long-term outcome in these patients and warrants more vigilance in future investigations

The NFKB1\it NFKB1 promoter polymorphism (-94ins/delATTG) is associated with susceptibility to cytomegalovirus infection after kidney transplantation and should have implications on CMV prophylaxis regimens

Infections with cytomegalovirus (CMV) are one of the most frequent opportunistic infections in kidney transplant recipients. Current risk-adapted CMV chemoprophylaxis regimens are based almost solely on the donor and recipient CMV serostatus. Of note, the $\it NFKB1$ -94ins/delATTG promoter polymorphism was recently associated with a higher risk of CMV infection. Since single genetic association studies suffer from poor reliability for drawing therapeutic implications, we performed this confirmatory study and included 256 kidney transplant recipients from 2007 to 2014 in this retrospective study. Patients were genotyped for the -94ins/delATTG $\it NFKB1$ promoter polymorphism and followed up for 12 months. The incidence of CMV infection within 12 months after kidney transplantation was 37.5% (33/88) for the ins/ins, 21.5% (28/130) for the ins/del, and 23.7% (9/38) for the del/del genotypes ($\it p$ = 0.023). Moreover, we evaluated the time of CMV infection onset. Ins/ins carriers had primarily late-onset CMV infection (median 194 days; interquartile range (IQR) 117–267 days) compared with heterozygous (ins/del; median 158 days; IQR 82–195 days) and homozygous deletion allele carriers (del/del; median 95 days; 84–123 days). Multivariate-restricted Cox regression model confirmed the ins/ins genotype to be an independent risk factor for the development of late-onset CMV infections. These findings should have an impact on post-kidney transplantation CMV chemoprophylaxis regimens

DNA methylation of a NF-κ\kappaB 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

The pro-inflammatory deletion allele of the NF-κ\kappaB1 polymorphism is characterized by a depletion of subunit p50 in sepsis

The functionally important NF-$\kappa$B1 promoter polymorphism (−94ins/delATTG) significantly shapes inflammation and impacts the outcome of sepsis. However, exploratory studies elucidating the molecular link of this genotype-dependent pattern are lacking. Accordingly, we analyzed lipopolysaccharide-stimulated peripheral blood mononuclear cells from both healthy volunteers ($\it n$ = 20) and septic patients ($\it n$ = 10). All individuals were genotyped for the −94ins/delATTG NF-$\kappa$B1 promoter polymorphism. We found a diminished nuclear activity of the NF-$\kappa$B subunit p50 in ID/DD genotypes after 48 h of lipopolysaccharide stimulation compared to II genotypes ($\it p$ = 0.025). This was associated with higher TNF-α (p = 0.005) and interleukin 6 concentrations (p = 0.014) and an increased production of mitochondrial radical oxygen species in ID/DD genotypes ($\it p$ = 0.001). Although ID/DD genotypes showed enhanced activation of mitochondrial biogenesis, they still had a significantly diminished cellular ATP content ($\it p$ = 0.046) and lower mtDNA copy numbers ($\it p$ = 0.010) compared to II genotypes. Strikingly, these findings were mirrored in peripheral blood mononuclear cells taken from septic patients. Our results emphasize the crucial aspect of considering NF-$\kappa$B subunits in sepsis. We showed here that the deletion allele of the NF-$\kappa$B1 (−94ins/delATTG) polymorphism was associated with the lower nuclear activity of subunit p50, which, in turn, was associated with aggravated inflammation and mitochondrial dysfunction

A novel understanding of postoperative complications

$\bf Background$ Propofol is a widely used anaesthetic drug with advantageous operating conditions and recovery profile. However, propofol could have long term effects on neuronal cells and is associated with post-operative delirium (POD). In this context, one of the contributing factors to the pathogenesis of POD is a reduction of cholinesterase activity. Accordingly, we investigated the effects of propofol on the methylation, expression and activity of cholinergic genes and proteins in an $\it in-vitro$ model. $\bf Results$ We found that propofol indeed reduced the activity of AChE / BChE in our $\it in-vitro$ model, without affecting the protein levels. Furthermore, we could show that propofol reduced the methylation of a repressor region of the CHRNA7 gene without changing the secretion of pro–or anti-inflammatory cytokines. Lastly, propofol changed the expression patterns of genes responsible for maintaining the epigenetic status of the cell and accordingly reduced the tri-methylation of H3 K27. $\bf Conclusion$ In conclusion we found a possible functional link between propofol treatment and POD, due to a reduced cholinergic activity. In addition to this, propofol changed the expression of different maintenance genes of the epigenome that also affected histone methylation. Thus, propofol treatment may also induce strong, long lasting changes in the brain by potentially altering the epigenetic landscape

Methazolamide reduces the AQP5 mRNA expression and immune cell migration

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 $\it (Aqp5)$ knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide ($10^{−5}$ M) and furosemide ($10^{−6}$ M) reduced $\it AQP5$ mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-$\alpha$ compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced $\it AQP5$ expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce $\it AQP5$ expression and migration of immune cells. However, after LPS administration, the reduction in $\it AQP5$ expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing $\it AQP5$ expression and has the potential to be used in sepsis prophylaxis