Study design and population
The present study was conducted in accordance with the Declaration of Helsinki (2013 edition) adopted by the World Medical Association [22]. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (PJ-KY-2018-12), Dalian, China. A written informed consent was obtained from all participants or legal guardians.
A total of 135 consecutive septic patients combined with acute kidney injury, who were admitted to the emergency ICU of our hospital from September 2016 to December 2018 and had the indication of CVVH treatment, were selected as subjects. The enrolled patients with sepsis were randomly divided into two groups: CVVH group, patients who received CVVH treatment; conventional treatment (non-CVVH) group. A total of 21 healthy volunteers were enrolled.
Inclusion and exclusion criteria
Patients with sepsis or septic shock were included in the present study if they met the requirement of the third international consensus definitions for sepsis and septic shock (Sepsis-3), accompanied by sepsis-induced acute kidney injury (diagnosed according to the KDIGO clinical practice guidelines) with increase in serum creatinine of more than 300 μmol/L, oliguria (<100 mL/6 h) unresponsive to fluid resuscitation, severe acidemia of less than pH of 7.2, hyperkalemia of more than 6.5 mmol/L, or clinically significant organ edema (e.g., pulmonary edema) [1, 11, 23-25].
Patients were excluded from the present study based on the following criteria: patients < 18 years old; patients with immunodeficiency virus infection, neutropenia (defined as an absolute neutrophil count of < 1,000 neutrophils/μL), acute bleeding, malignant tumor, pregnancy, or chronic kidney disease; patients who used corticosteroids or nephrotoxic drugs; patients who received any kind of renal replacement therapy prior to admission to the ICU; patients who refused to take part in the present study.
Treatments of the patients
The conventional therapy was based on standard sepsis treatments and according to Surviving Sepsis Campaign guidelines, which included intensive monitoring, fluid resuscitation, oxygen administration or mechanical ventilation, antimicrobial therapy (blood culture was conducted before the administration of antibiotics), vasopressor administration, glucose control, diuretics for oliguria with fluid overload, etc [3].
For the CVVH treatment, a 11.5Fr double lumen hemofiltration catheter (Lily Technology, Guangdong, China) was percutaneously inserted into the femoral vein. Hemofiltration was performed using the PrimaFlex apparatus (ALPRI, France) equipped with an acrylonitrile and sodium methallyl sulfonate copolymer + polyethylene (AN69ST) hollow-fiber high-flux hemofilter (1.5 m2, Braun Diapact, Germany), and was delivered in CVVH mode. The extracorporeal circuit and hemofilter were pre-flushed with heparinized normal saline (12500 U of unfractionated heparin added in 1000 mL of normal saline) to prevent clotting. After circulation, the heparinized normal saline was flushed out of the hemofilter and circuit tubes prior to starting CVVH. Continuous intravenous pumping of unfractionated heparin (4 to 8 U/kg/h) was used to adjust the systemic activated partial thromboplastin time (APTT) between 35 and 45 s [26]. However, heparin-free CVVH was implemented if a patient had a bleeding tendency (e.g. low platelet count, prolonged activated partial thromboplastin time). CVVH was initiated within 12 h of ICU admission and maintained for at least 72 h with the doses of 35 ~ 60 mL/kg/h, and a blood flow rate of 150 ~ 200 mL/min. CVVH were stopped on basis of renal recovery and physician decision [24] The hemofilter was used till 72 h, or immediately changed if it did not work effectively because of clotting. The bicarbonate replacement solutions were commercially prepared by Qingshan Likang, Pharmaceutical Co. (Chengdu, China), and was pre-hemofilter diluted.
Variables
The following variables were collected for each patient on admission: age, sex, infection sites, results of blood culture, laboratory data (plasma creatinine, lactate, procalcitonin, leukocyte count, hemoglobin, platelet count and C-reactive protein), mean arterial pressure, number of patients using vasopressors, time from sepsis onset to ICU admission and number of patients using mechanical ventilation. On days 1, 3 and 7 of ICU admission, CD4+ T lymphocyte subsets (Th1, Th2, Th17 and Treg) were measured, and Th1/Th2 and Th17/Treg ratios were calculated; meantime, sequential organ failure assessment (SOFA) scores were evaluated.
Flow cytometry analysis
Two mL of peripheral venous blood was taken into EDTA-K3 coated tubes. The blood sample was first stimulated for 5 h with a cell stimulation cocktail (Sigma, St. Louis, MO, USA), containing 50 ng/mL of protein transport inhibitors, 1 mg/mL of ionomycin and 1.7 mg/mL of monensin, according to manufacturer’s instructions.
T lymphocyte subsets, including Th1 (CD3+CD8-IFN-γ+), Th2 (CD3+CD8-IL-4+), Th17 (CD4+IL-17+) and Treg (CD4+CD25+Fox3+), were real-timely measured by flow cytometry (FACSVantage SE, BD Biosciences, San Jose, CA, USA). For the Th1 and Th2 analysis, the whole blood cells were extracellularly stained with FITC anti-human CD8 and PE-cy5 anti-human CD3 (BD Biosciences, San Jose, CA, USA) at 4°C for 30 min to determine the T lymphocyte population. Afterwards, the red blood cells in the whole blood were lysed using the FACS lysing solution (BD Biosciences, San Jose, CA, USA), and centrifuged (2,000×g) for five minutes. Then, the supernatant was abandoned, and the cell pellet was washed with phosphate buffered saline (PBS) and permeabilized with the Fix & Perm Reagent (BD Biosciences, San Jose, CA, USA). The permeabilized cells were further intracellular stained with either PE anti-human INF-γ antibody (Abcam, Cambridge, MA, USA) for the detection of Th1 cells (Fig.1), or PE anti-human IL-4 antibody (Abcam, Cambridge, MA, USA) for the detection of Th2 cells (Fig.1).
For the Th17 and Treg analysis, the stimulated peripheral venous blood was extracellularly stained with the following combinations of antibodies: PE-cy5-conjugated anti-CD4 (Abcam, Cambridge, MA, USA) and PE anti-human IL-17 (BD Biosciences, San Jose, CA, USA) for Th17 (Fig.1); PE-cy5-conjugated anti-CD4 (Abcam, Cambridge, MA, USA), FITC-conjugated anti-CD25, and PE-conjugated anti-Fox3 (Abcam, Cambridge, MA, USA) for Treg (Fig.1).
Statistical analysis
Data were analyzed using SPSS 22.0 (IBM, Armonk, NY, USA). Data were expressed as mean ± standard deviation (SD) for normal distribution, or median and interquartile range for skewed distribution. Pearson’s chi-squared or Fisher’s exact test was used, as appropriate, to compare the categorical variables. A Mann-Whitney U test was used to compare the variables between the CVVH and non-CVVH groups. Variables among time points and groups were compared using repeated-measures analysis of variance (ANOVA), followed by Bonferroni test for multiple comparisons. The differences were considered statistically significant when P < 0.05.