CCL-2 and CXCL-8: Potential Prognostic Biomarkers of Acute Kidney Injury after a Bothrops atrox Snakebite

Abstract

In the Brazilian Amazon, the snake Bothrops atrox is the primary cause of snakebites. B. atrox (BaV) venom can cause systemic pathophysiological changes such as acute kidney injury (AKI), which leads to the production of chemokines and cytokines in response to the envenomation. These soluble immunological molecules act by modulating the inflammatory response; however, the mechanisms associated with the development of AKI are still poorly understood. Here, we characterize the profile of these soluble immunological molecules as possible predictive biomarkers of the development of AKI. The study involved 34 patients who had been victims of snakebites by Bothrops sp. These were categorized into two groups according to the development of AKI (AKI^(-)/AKI⁽⁺⁾), using healthy donors as the control (HD). Peripheral blood samples were collected at three-time points: before antivenom administration (T0) and at 24 and 48 hours after antivenom (T1 and T2, respectively). The soluble immunological molecules (CXCL-8, CCL-5, CXCL-9, CCL-2, CXCL-10, IL-6, TNF, IL-2, IL-10, IFN-γ, IL-4, and IL-17A) were quantified using cytometric bead array. Our results demonstrated an increase in CXCL-9, CXCL-10, IL-6, IL-2, IL-10, and IL-17A molecules in the groups of patients who suffered Bothrops snakebites (AKI^(-) and AKI⁽⁺⁾) before antivenom administration, when compared to HD. In the AKI⁽⁺⁾ group, levels of CXCL-8 and CCL-2 molecules were elevated on admission and progressively decreased during the clinical evolution of patients after antivenom administration. In addition, in the signature analysis, these were produced exclusively by the group AKI⁽⁺⁾ at T0. Thus, these chemokines may be related to the initiation and extension of AKI after envenomation by Bothrops and present themselves as two potential biomarkers of AKI at T0.

Figure 1

Flowchart of study. Thirty-four patients were considered eligible and were divided into 2 groups according to clinical evolution: without acute kidney injury (AKI^(-)) and with acute kidney injury (AKI⁽⁺⁾). The immunological molecules were quantified using the CBA (cytometric bead array) technique before antivenom administration (T0) and after antivenom administration (T1-24 h and T2-48 h).

Figure 2

Concentration of soluble immunological molecules on admission of the patient (T0), in the groups AKI⁽⁺⁾, AKI^(-), and HD. ^∗p < 0.01;  ^∗∗p < 0.008;  ^∗∗∗p < 0.0001. The results are expressed as the mean ± standard deviation in MFI (mean fluorescent intensity), and the statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's test.

Figure 3

Ascendant signature of soluble immunological molecules presented by AKI(-) and AKI(+) patients. Performed based on the global median of each of the dosed cytokines and chemokines, using data from all patients (HD (healthy donors), AKI^(-) without acute kidney injury, and AKI⁽⁺⁾ with acute kidney injury). The global median of each molecule was used as a cut-off point, expressed in MFI (mean fluorescent intensity), thus classifying the individual as a “low” or “high” producer of the dosed molecules. Statistical significance was considered in all cases at p < 0.05. In the Venn diagram, it is possible to identify which molecules prove to be potential biomarkers because they are present in high concentrations only in the AKI⁽⁺⁾ group.

Figure 4

Analysis of the dynamics of soluble immunological molecule production during the clinical evolution of patients. In the background, the interquartile range [25–75] of the concentration of cytokines and chemokines in the HD healthy donors group is used as a baseline. The statistical difference between the groups AKI⁽⁺⁾ with acute kidney injury and AKI^(-) without acute kidney injury was considered when p < 0.05 (┌┐). The results are expressed as the mean ± standard deviation in MFI (mean fluorescent intensity), and statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's posttest.

Figure 5

Network of soluble immunological molecules shows interactions occurring between groups in the follow-up of the study. Each color group is used to identify chemokines (yellow box) and cytokines (orange box). Dashed lines between molecules indicate a negative correlation while solid lines indicate a positive correlation. The thickness of these indicates the strength of the correlation. The correlation index (r) was used to categorize the strength of the correlation as either weak (r ≤ 0.35), moderate (r ≥ 0.36 to r ≤ 0.67) or strong (r ≥ 0.68).

Figure 6

Probable pathogenic mechanism for the development of acute kidney injury after a B. atrox snakebite. Acute kidney injury initially results from the direct damage by the venom to the renal vascular endothelium. After activation of the endothelium, renal epithelial cells, tubular cells, and others produce chemotactic molecules, such as CCL-2 and CXCL-8, and synergistically increase the expression of p-selectins and cell adhesion molecule 1 (ICAM-1). Neutrophils and monocytes adhere to endothelial cells and migrate through the interstitium, causing changes in vascular permeability and compromising the cell integrity of the endothelium and renal tubules. A capillary plug is formed with the infiltration of neutrophils, monocytes, platelets, and red blood cells. Infiltrated neutrophils and monocytes release proinflammatory chemokines and cytokines, exacerbating the immune response, followed by alterations such as NTA and cell apoptosis that lead to decreased renal function with accumulation of metabolites and electrolytes in the body, resulting in acute renal failure in these patients. It is noteworthy that the cells represented in the figure were not evaluated in the present study.