Evaluation of the Effectiveness of Continuous Venovenous Hemodiafiltration Appli̇ed With Oxiris and AN69 Membranes in Patients With Septic Shock-Related Acute Kidney Injury

Background and objectives: AN69 and Oxiris are filters used in continuous renal replacement therapy. In this study, we aimed to research the effects of these filters on blood cell counts, blood biochemistry, inflammation indicators, clinical status and mortality of patients diagnosed with septic shock-related acute kidney injury. Method: Between March 2019 and October 2019, 42 adult patients (Group 1: Oxiris (n = 21) or Group 2: AN69 (n=21)) with septic shock-related acute kidney injury and received continuous venovenous hemodiafiltration (CVVHDF) in the intensive care unit were included in the study and their results were prospectively observed and compared. The data at the begining of CVVHDF (pre-CVVHDF) and 24 hours after the onset of CVVHDF (post-CVVHDF) were recorded. Results: In the comparison of the pre- and post-CVVHDF values in Group 1, there was a statistically significant decrease detected in the procalcitonin (p = 0.04) and noradrenaline infusion rate (p = 0.02) levels. In terms of the other data there was no statistically significant difference between pre- and post-CVVHDF values in Group 1. In the comparison of the pre- and post-CVVHDF values in Group 2, there was a statistically significant decrease detected in the urea (p = 0.04), platelet count (p = 0.02) and procalcitonin (p = 0.002) levels. There was no statistically significant difference between pre- and post-CVVHDF values in terms of the other data in Group 2. There was no statistically significant difference between the groups in terms of mortality. Conclusions: CVVHDF with Oxiris filter causes a statistically significant decrease in noradrenaline infusion rate. Therefore, we think that the use of CVVHDF with Oxiris filter applied for septic shock-related acute kidney injury will save us time and increase the improvement in the treatment.


Introduction
Septic shock is a subset of sepsis with circulatory, cellular and metabolic abnormalities.
Patients with septic shock may be clinically defined by the need for vasopressors to maintain mean arterial pressure ≥65 mmHg and serum lactate levels> 2 mmol / L (> 18 mg / dL) in the absence of hypovolemia (1).
Lipoteichoic acid (LTA) in the structure of gram-positive bacteria and lipopolysaccharide (LPS) -endotoxin-in the structure of gram-negative bacteria are proinflammatory bacterial lipids. These bacterial lipids induce proinflammatory cytokine synthesis by signaling monocytes, macrophages, neutrophils and other immune cell types (2). Proinflammatory cytokines induce a cytokine storm, causing endothelial cell disorder. Large molecules and liquid are extravasated into the interstitium (3). As a result, organ dysfunction develops (4). One of the organ dysfunctions that may develop in sepsis is Acute Kidney Injury (AKI).
In septic AKI, microcirculatory disorder develops in renal parenchyma as a result of deregulation of inflammatory mediators, immune cell infiltration and nitric oxide synthase (5).
Treatment of AKI involves treating the underlying disease and supporting renal function with renal replacement therapy (RRT). However, The Surviving Sepsis Campaign Guidelines (6) contain little explanation regarding AKI treatment.
Many studies, with conflicting results, have evaluated the capacity of extracorporeal devices to adsorb endotoxins and cytokines (7,8), but The Surviving Sepsis Campaign Guidelines (6) have not made any recommendations on the use of blood purification techniques.
The size of the endotoxin molecules is about 10 kDa. However, it can form aggregates up to 1,000 kDa consisting of covalently bound lipid and polysaccharide (9). Cytokines are molecules that dissolve in water and are in free form in circulation. Their molecular weight is between 0.5-60 kDa (10). RRT filters are semi-permeable membranes with approximately 35 kDa pores (11). Two of these filters, AN69 (M100, Gambro, France) and Oxiris (Baxter, France), are widely used.
AN69 adsorbs cytokines but does not adsorb endotoxins (12). Oxiris adsorbs endotoxins (negatively charged) thanks to the positive charge on the surface in addition to cytokine elimination (13). Most studies of Oxiris include patients with sepsis/septic shock due to gram-negative bacteria. Because endotoxins are a component of gram-negative bacteria, rather than gram-positive bacteria. However, Oxiris may also be useful in the case of sepsis/septic shock due to gram-positive bacteria, as intestinal hypoperfusion usually causes gram-negative bacteria to pass through the digestive lumen into the blood (14).
We think that the use of Oxiris filter in patients with septic shock-related AKI will improve hemodynamics to save time for treatment, reducing the need for noradrenaline.
In this study, we aimed to research the effects of AN69 and Oxiris membranes on blood (Universität Kiel, Germany) was used to calculate the sample size with reference to the proportions specified in a previous study (4). The minimum number of patients to be included in the study was 42 with a two-tailed alpha error of 0.05, a power of 0.80, an allocation ratio of N2 / N1 = 1 and an effect size of 0.8. Forty-two patients with septic shock in our intensive care unit (1) who underwent continuous venovenous hemodiafiltration (CVVHDF) due to AKI (15) were included in the study and the results were compared.

Inclusion criteria
Age ≥18 years Presence of septic shock (1)

Exclusion criteria
Documented Stage 5 chronic kidney disease (glomerular filtration rate (GFR) <15 mL / min / 1.73 m2) End-stage renal failure in long-term dialysis Patients receiving RRT before admission to the ICU Patients with an inferior vena cava collabability index (IVCCI) that cannot be measured or have comorbidities that may affect outcomes (16).
Electrocardiography (ECG), pulse oximetry (SpO2) assessment and continuous invasive arterial pressure measurement after intraarterial cannulation were performed in all patients using the BSM-9101K monitor (Nihon Kohden Europe GmbH, Germany).
For vascular access, a double-lumen hemodialysis catheter (11.5 Fr, Scw medicath, China) was inserted into the femoral or internal jugular vein with ultrasonography (USG) guidance (GE Vivid device, United Medical Instruments, USA). In addition to standard treatment, all patients received CVVHDF for 24 hours using Prismaflex CRRT system (Gambro, Sweden) with the adsorbing Oxiris or AN69 filter. Daily dialysis dose was maintained between 35-45 mL / kg / h, blood flow between 100-150 mL / min, and filtration fraction was between 35-45%. DIALISAN CVVHD BG 2D (Baxter, Italy) was used as dialysis and postdilution solution.
For the anticoagulation of the circuit, continuous infusion of nonfractionated heparin was used in patients with hemorrhagic profile at physiological margin, and citrat solution was used in patients whose hemorrhage profile was not at physiological margin and / or who had bleeding risk. Heparin was used with an infusion rate of 5-15 IU / kg / h. The dosage of heparin was set for 45-60 seconds with activated partial thromboplastin time [aPTT] (ACL TOP500 and ACL TOP700, Instrumentation Laboratory, Bedford, MA, USA).
The researchers who made the diagnosis, applied the CRTT, calculated the USG and IVCCI, collected and evaluated the results were different. The data were collected from the electronic medical record system and patient files of our institution.
Demographic data of the patients, Acute Physiology and Chronic Health Interrogation (APACHE) II score, number of organs with failure, mortality after ARF, duration of ICU stay, source of sepsis, isolated microorganisms in blood culture, KDIGO stage, the type of filter used (AN69 or Oxiris), anticoagulation method (heparin / citrate) was recorded. Sequential Organ Failure Assessment (SOFA) score were recorded at the beginning of CVVHDF (pre-CVVHDF) and 24 hours after the onset of CVVHDF (post-CVVHDF). While writing the article, necessary checks were made with strobe statement checklist used for observational studies.

Statistical Analysis
SPSS 16.0 for Windows program was used for statistical analysis. Statistical data were expressed as mean and standard deviation, and categorical data were expressed as frequency and percentage. The comparison of categorical data in the groups was made with chi-square test and the results were given as n%. The Kolmogorov-Smirnov test was used to determine whether the numerical data matched the normality distribution.
Student's T-test was used for the evaluation of the numerical data matching the normal distribution between the groups, and Mann-Whitney U test was used for the non-normal distribution. Paired T-test and Wilcoxon Signed Rank test were used for comparison of two normal distribution measurements. Results regarding numerical data were given as mean ± standard deviation. P <0.05 was accepted as statistically significant.

Results
The mean age of the patients included in the study was 61.33 ± 20.01; Apache II scores were 29.26 ± 7.75 and SOFA 1 scores were 10,07 ± 2,78. At least 2 and at most 4 organ failure were detected in the patients included in the study. Fifty percent of the patients were female and 50% were male. During the study period twenty-five of the patients were died. Mortality rate was 59.5%. Heparin was used as anticoagulant in 81% (n = 34) and citrate was used in 19% (n = 8). Twenty-one patients had KDIGO stage 3, 13 had KDIGO stage 2, and 8 had KDIGO stage 1. There was no statistically significant difference between the two groups in terms of KDIGO stages. The most common source of sepsis was the lungs (33.3%). The distribution of the patients in terms of the source of sepsis is shown in Figure 1.
The source of sepsis, reproduction in blood culture and the type of filter used are shown in Table 1. Among the gram-negative bacteria P. aeruginosa was found in the blood culture of 7 patients, A. Baumannii in 7, K. Pneumoniae in 4 and Enterobacter spp. in 3 patients; while among the gram-positive bacteria S. epidermidis was found in the blood culture of 3 patients, S. aureus in 3 and S. hominis ssp hominis in 3 patients (Table 1).
In 21 patients, CVVHDF was performed using Oxiris filter (Group 1) and in 21 patients CVVHDF was performed using AN69 filter (Group 2). When the blood cultures of the patients using Oxiris filter were examined 12 patients had gram-negative, 6 patients had gram-positive bacteria growth and 3 patients had no growth. When the blood cultures of the patients using AN69 filter were examined 11 patients had gram-negative, 6 patients had gram-positive bacteria growth and 4 patients had no growth. There was no statistically significant difference between the groups in terms of gram-negative and gram-positive bacteria growth in blood culture ( Table 2).
The comparison of the groups in terms of clinical characteristics and laboratory values is shown in Tables 3 and 4. There was no statistically significant difference between the groups in terms of clinical features and laboratory values.
In the comparison of the pre-and post-CVVHDF values in Group 1, there was a statistically significant decrease detected in the procalcitonin (p = 0.04) and noradrenaline infusion rate (p = 0.02) levels. In terms of the other data there was no statistically significant difference between pre-and post-CVVHDF values in Group 1. In the comparison of the pre-and post-CVVHDF values in Group 2, there was a statistically significant decrease detected in the urea (p = 0.04), platelet count (p = 0.02) and procalcitonin (p = 0.002) levels.
There was no statistically significant difference between pre-and post-CVVHDF values in terms of the other data in Group 2 (Table 4). Table 5 shows the comparison of the groups in terms of gender, mortality and anticoagulant used. There was no statistically significant difference between the groups in terms of gender, mortality and anticoagulants used.

Discussion
In our study, at the end of the CVVHDF for 24 hours we detected that PRC and NIR were reduced in patients using Oxiris filter; urea, platelet count and PRC levels were decreased significantly in patients using AN69 filter. There was no statistically significant difference between the groups in terms of mortality. Damaged host cells express surface damage-associated molecular patterns (DAMPs) such as high-mobility-group-box-1 protein (HMGB1) on their surface. DAMPs can be released into circulation and are recognized by pattern recognition receptors (PRRs). Thus, leukocyte activation and cytokine synthesis are increased, fueling the vicious cycle of uncontrolled immuno-inflammatory process. Excessive release of cytokines in the blood is defined as a "cytokine storm" (14). As a result, sepsis or septic shock develops through vasodilatation, endothelial leakage and organ dysfunction (4). One of the organ dysfunctions in sepsis is AKI. Although the pathophysiological mechanism in septic AKI is not fully understood, it is clear that the inflammatory cascade of sepsis contributes to AKI (19). The basic pathophysiological paradigm correlates septic AKI with decreased global renal blood flow, secondary tubular epithelial cell death, or acute tubular necrosis. The reason for this belief is that AKI is associated with hypoperfusion and shock, and that ischemic damage can lead to intense cell death (eg acute tubular necrosis). However, the importance of ischemia-reperfusion is increasing (20).
Septic shock is associated with higher mortality compared to sepsis (1). Septic shock continues to account for 62% of general deaths and hospital mortality rates are above 40% (21). Mortality associated with AKI is high (40-60%) and the short-and long-term outcomes in the form of chronic or end-stage renal disease are devastating (5). AKI develops in more than 45% of patients with septic shock (21). The mortality rate of our study patients was 59.5%. Considering the fact that our patient group had septic shockrelated AKI, our mortality rate was within normal limits. There was no significant difference between the groups in terms of mortality.

The Sepsis Occurrence in Acutely Ill Patients (SOAP) study reported an almost equal
prevalence of gram-positive and gram-negative bacterial infections in patients with sepsis (24). Although subsequent studies (26) have suggested an increase in the incidence of gram-positive organisms, The 2012 Intensive Care Over Nations (ICON) study has shown that gram-negative bacterial infections are more common in the United States than grampositive bacterial infections (27). In our study, gram-negative bacteria grew more in the blood cultures of the patients than gram-positive bacteria.
In a study of 13796 infected ICU patients published in 2009 S. aureus, Pseudomonas species, Enterobacteriaceae (especially E. coli) and fungi were the most common blood cultures. In this study, Acinetobacter accounted for 9% of patients with positive blood culture (25). In a review published in 2012, the most common isolated gram-negative bacteria were E. coli, P. aeruginosa and K. pneumonia; and and the most common grampositive bacteria were S. aureus, S. pneumonia and Enterococcus spp. in patients with sepsis and septic shock (28). In our study, the most common gram-negative bacteria were P. aeruginosa, A. Baumannii, K. Pneumoniae and Enterobacter spp; and the most common gram-positive bacteria were S. epidermidis, S. aureus and S. hominis ssp hominis in the blood culture. Our study was consistent with previous studies in terms of blood culture reproduction. However, 20% of patients with positive blood culture had acinetobacter reproduction and this rate was higher than previous studies.
Continuous renal replacement therapy (CRRT) with improvement in extracorporeal blood purification techniques and membrane materials are widely used in critical illness (10).
The theoretical cut-off for an RRT membrane is about 35 kDa. Throughout this membrane, diffusion and convection can take place. Diffusion follows a concentration gradient (as in intermittent hemodialysis) and is an ideal method to remove small (<500 Da) molecules such as creatinine. Convection follows a hydrostatic pressure gradient and is the best method for elimination of medium to large (500 Da to 60 kDa, 13,750 Da beta-2 microglobulin) and large molecules (60 to 100 kDa, eg 70 kDa albumin). In clinical practice, high (60 kDa) or median (50 kDa) cut-off membranes are almost never used because high cut-off membranes may increase the risk of albumin loss (11). Endotoxin molecules have a size of approximately 10 kDa, but can form aggregates of up to 1,000 kDa consisting of a covalently bonded lipid and polysaccharide (9). The smaller molecular weight of the cytokines, the more cytokines will be removed in the CRRT. AN69 is a copolymer of hydrophobic acrylonitrile and hydrophilic sodium metahalylsulfonate. As AN69 is negatively charged due to sulfonate groups, the AN69 membrane adsorbs cytokines via ionic bonding between its sulfonate group and the amino group on the surface of a cytokine molecule (12). Oxiris is a high permeability polyacrylonitrile (AN69) based membrane on which a surface treatment of a positively charged polyethylene is added onto the hemofilter. Thanks to this positive charge on the surface of the Oxiris filter -in addition to the bulk cytokine elimination in the mass of the membrane -endotoxin (negatively charged) adsorption takes place (13). In in-vitro studies, Oxiris filter emerges as a hemoperfusion device capable of eliminating both endotoxin and cytokine (29). Endotoxin hemoadsorption can reduce the pathogenic activity and organ dysfunction of endotoxin. Cytokine removal by hemofiltration or hemoadsorption can restore the status of immune homeostasis. It is thought that the use of semi-permeable membranes that can provide endotoxin and cytokine elimination is a valuable treatment option in septic shock due to gram-negative bacterial infection (30).
Endotoxin may also be present in the circulation due to translocation from the ischemic gut in gram-positive infections (4). Therefore, the use of oxiris in gram-positive sepsis or septic shock may be beneficial (14).
The main finding of our study is that CVVHDF performed with Oxiris filter improves hemodynamics, reduces NIR, is clinically applicable and has no side effects in patients with septic shock-related AKI. Our results confirm the results of some studies in which Oxiris filter was applied in the same patient group. Comparing Oxiris and AN69 filters in patients with septic shock-related AKI Broman et al. found a strong decrease in circulating endotoxin and cytokine levels as a result of CVVHDF treatment with Oxiris filter. This reduction was associated with a favorable hemodynamic effect, such as a faster decrease in blood lactate levels and a decrease in NIR required to maintain mean arterial pressure.
There was a blunted cytokine response in both filter groups, but the decrease in TNF-α, IL-6, IL-8, and IFN-γ was more outstanding in patients treated with Oxiris than the AN69 filter (4). In the study of Schwindenhammer et al., 31 patients were diagnosed with septic shock between 2014 and 2019 and one of the continuous venovenous hemofiltration (CVVH) or CVVHDF therapies with Oxiris filter was applied. A relative decrease of 88% was observed in NIR. Lactatemia and pH improved significantly over time (13). In their study on 60 septic patients published in 2019, Turani et al. found that CVVHDF with Oxiris filter improved basic cardiorenal and respiratory parameters and decreased NIR (31).
Shum et al., in a study performed CVVH for 48 hours, found a 37% reduction in the SOFA score of sepsis-related AKI patients who underwent Oxiris filter compared to polysulfone-based standard filter (30). In their study published in 2019, Turani et al. found that the SOFA score of 60 septic patients applied CVVHDF with Oxiris filter decreased from 12.4 ± 2 to 9 ± 2 (31). In our study, no significant decrease was observed in the SOFA score by CVVHDF with Oxiris filter. This is due to the fact that our patients had very severe diseases with high mortality (septic shock-related AKI) and we evaluated the SOFA score after 24 hours of Oxiris administration. Longer CRRT could cause a significant decrease in SOFA score.
In their study on 13 patients with sepsis and multiorgan failure Dahaba (31). In our study, we found that PRC levels decreased significantly in both groups using Oxiris and AN69 filters after 24 hours of CVVHDF. The cut-off value of AN69 filter is 35-40 kDa (32) and PCT molecular weight is 14.5 kDa (33). We attributed the significant decrease in PCT value after CVVHDF with both filters to the fact that the molecular weight of PCT was considerably lower than the cut-off value.
The limitation of our study is that we evaluate the blood cell counts, blood biochemistry, inflammation indicators, clinical conditions and the mortality results without considering other intermittent conditions after the 24 hours of CVVHDF. We compared the changes that occurred with only one CVVHDF application. In the future, randomized, controlled, double-blind, clinical trials can be planned to compare the changes in renal function and mortality rates of CVVHDF with longer or repeated administration.

Conclusions
CVVHDF with Oxiris filter leads to a statistically significant decrease in NIR. Therefore, we think that the use of CVVHDF with Oxiris filter applied for septic shock-related AKI will save us time and increase the improvement.

3.Availability of data and materials:
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. (akyektas@hotmail.com)

4.Competing interests:
The authors declare no competing interests.

6.Author contribution:
AKY, OU and CKK carried out the anterior and posterior sciatic nerve block, participated in the sequence alignment and drafted the manuscript, participated in the design of the study and performed the statistical analysis. DK and EY conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.   Figure 1 Classification of patients in terms of the source of sepsis