Elevated Levels of Interleukin-21 in Acute Kidney Injury After Cardiopulmonary Bypass Surgery

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Objective
However, to our knowledge, there is no published study relating to the IL-21 and AKI-CPB. Therefore, this study was performed to determine the role of IL-21 in AKI development after heart surgery.

Materials and Methods Population
Overall, 90 patients undergoing CPB graft surgery in Shahid Mohammadi Hospital, Bandar Abbas, Iran (from January 2017 to September 2018) were enrolled in this experimental before-after study. After surgery, 40 patients developed AKI. Patients were classified according to the AKI Network (AKIN) definition (22); an increase of 0.3 mg/dL in the serum creatinine level up to 48 hours post operation is defined as AKI. This group was compared to non-AKI subjects (n = 50). Patients were excluded if they were diagnosed with autoimmune and infectious diseases, any kind of liver malfunctions and kidney disorders or transplants, hemodialysis, inflammatory disease, and pre-existing AKI. Addicted patients (except for cigarettes), patients undergoing coronary angiography 72 hours before sample collection, and patients medicated with immunosuppressive or nephrotoxic drugs were also excluded from the study. All included subjects signed written informed consent forms, and systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥90 mm Hg was defined as hypertension (23). Body weight in kilograms divided by height in meters squared was defined as the body mass index (BMI = kg/m 2 ). Subjects who smoked at least 2 cigarettes a day at the time of admission were defined as smokers.

Blood Samples and Data Collection
Before surgery and 12 and 48 hours after the operation, blood was obtained from patients. For the collection of serum samples, blood was coagulated at room temperature for 30 minutes and then underwent centrifugation (2600 g, 10 minutes, Sigma 2-16KL). Serum creatinine levels were evaluated before and 48 hours post-operation using the Cobas 700 analyzer (Roche Diagnostics, USA). Demographic (including age, gender, and smoking) and operation (e.g., CPB time, cross-clamp time, and ejection fraction) variables were obtained from participants' medical records.

Serum IL-21 Evaluation
The IL-21 serum level was determined using an enzymelinked immunosorbent assay kit (Zell bio, Germany) before and 12 hours after surgery according to the provider's instructions. The IL-21 linear standard curve was developed using the standards provided by the manufacturer. The sensitivity of the kit and the intra and inter-assay variations were 25 pg/mL and < 10%, respectively.

Statistical Analyses
Quantitative variables, including demographic and clinical data, were compared between AKI and non-AKI groups using a t test or Mann-Whitney U-test if customarily or non-normally distributed, respectively, and presented as mean ± standard deviation (SD). Categorical variables were analyzed by the chi-square test. The association between IL-21 and clinical variables analyzed using Pearson correlation and correlation coefficient (r) was reported as well. The sample size was calculated considering a precision/absolute error of 5% and a type 1 error of 5%. The SD of variables was taken from previous studies. All statistical analyses were performed with the SPSS (version 20.0, SPSS Inc., Chicago, IL, USA), and statistical significance was defined as P < 0.05.

Clinical Characteristics and IL-21 Concentration in Study Participants
A total of 90 (40 AKI and 50 non-AKI) patients were included in this study. The severity of AKI was classified based on the AKIN criteria, and 12 (30%), 18 (45%), and 10 (25%) patients developed stages 1, 2, and 3, respectively. Clinical and operative variables are presented in Table 1. The mean age of patients in AKI and non-AKI groups was 60.1 ± 6.4 and 59.4 ± 5.1, respectively (P = 0.5). Eighteen men and 22 women developed AKI, and 25 men and 25 women were diagnosed as non-AKI cases. Other clinical and demographic variables were not significantly different between AKI and non-AKI subjects (P > 0.05). The IL-21 serum level was compared in CPB patients before and after the operation. A significant increase of IL-21 was observed 12 hours after operation compared to the amounts of this cytokine before surgery in both AKI (21.4 ± 6.3 and 15.7 ± 5.5, P < 0.001) and non-AKI (19.1 ± 6.4 and 13.7 ± 6.3, P < 0.001) participants. However, no significant difference was found between AKI and non-AKI subjects before or 12 hours after the operation ( Table 1).

Association of IL-21 and Other Variables
The correlation between IL-21 and other variables, including age, BMI, and serum creatinine levels before and 48 hours after the operation was assessed, and the results (Table 2) demonstrated no significant association between IL-21 and selected variables.

Discussion
This study evaluated the post-CPB IL-21 levels and their association with AKI development. Our findings showed that IL-21 levels were significantly increased after CPB. However, there was no association between IL-21 levels hmj.hums.ac.ir http and the incidence of AKI-CPB. Our results about the higher post-CPB levels of IL-21 are consistent with those of previous studies, confirming the elevated concentration of IL-21 in many inflammatory diseases such as asthma (24), airway allergic inflammation (25), inflammatory bowel disease (26), experimental autoimmune encephalitis, and (27) psoriasis (28). Our findings are also in line with those of studies that reported that mice deficient in IL-21 or IL-21R had reduced inflammatory responses following experimental colitis (29) and parasitic infections (30). Moreover, our results are consistent with the findings of previous studies, demonstrating a shift in the cytokine balance toward a predominant proinflammatory type after CPB (31). Although the exact mechanism of how the increased levels of IL-21A may affect the postoperative inflammatory response needs further investigations, there is some explanatory evidence in this regard. First, IL-21 is not only produced by Th17 cells but also induces IL-17 production via STAT3dependent mechanisms (11). The results of the study by Yang et al on human CD4 T cells represented that IL-21 accompanying transforming growth factor-beta has a role in the differentiation of naive T cells into Th17 cells (13), which are involved in the pathogenesis of post-CPB systemic inflammatory response syndrome (32). IL-21 also up-regulates the IL-23R expression necessary for the development of Th17 cells (14). Second, IL-21 decreases CD4 T cells' response to the suppressive effects of Treg cells, which can disturb immunologic homeostasis (33). Third, IL-21 plays a critical role in the differentiation of human B cells and immunoglobulin (Ig) production (34). It is noteworthy that the correlation of some antibodies (e.g., heart-reactive antibodies) with postcardiotomy syndrome in patients undergoing a procedure bypass has been reported previously (35).
Contrary to our assumptions, our data did not approve any association between increased IL-21 and CPB-AKI risk. This finding contradicts those of earlier studies that confirmed the effects of Th17-related cytokines on the development of renal injuries in kidney ischemiareperfusion injury, glomerulonephritis, and lupus nephritis (6)(7)(8). Our results did not support those of Herber et al, indicating that MRL-Faslpr mice treated with anti-IL-21R/Fc did not have thickening in glomerular basement membranes and revealed reduced levels of glomerular IgG deposits in the kidney (36). Our study had some limitations, including a relatively small sample size, thus caution is still appropriate in generalizing our result. The continuation of the study should allow us to give a more precise picture of the IL-21 effect on the CPB-AKI incidence. Further large prospective studies are recommended in this respect. Generally, it seems that IL-21 as a competent inflammatory mediator, not only exerts its effects through its own signaling but also via modulating other members of the IL-17 family, may contribute to systemic inflammatory responses after CPB. We also demonstrated that IL-21 could not be a biomarker in predicting renal outcomes in patients after CPB.

Acknowledgments
We extend our thanks to Miss. Gisoo Mehri for her helpful comments on our manuscript and the Research Council of Hormozgan University of Medical Sciences for financial support.

Authors' Contribution
NN developed the concept, and prepared the manuscript, MM performed experimentation, MR analyzed data, prepared figures and tables, wrote the method and result sections and carefully read the manuscript. HM and MKH prepared the samples. All authors