EIF2AK2 protein targeted activation of AIM2-mediated PANoptosis promotes sepsis-induced acute kidney injury

Abstract

Background: Acute kidney injury (AKI) frequently occurs as a complication of sepsis. PANoptosis refers to a type of inflammatory programmed cell death that exhibits key characteristics of apoptosis, necroptosis, and pyroptosis. Here, we evaluated the role of absent in melanoma 2 (AIM2) and eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) in septic AKI.

Methods: A septic AKI model was created through cecal ligation and puncture (CLP), while an in vitro model was developed using lipopolysaccharide (LPS)-stimulated HK2 cells. Hematoxylin and eosin (HE), Periodic acid-Schiff (PAS), and TUNEL staining were conducted to assess kidney injury in mice. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by kits. Gene expression was detected utilizing RT-qPCR, and Western blot was used to test protein levels. Immunofluorescence was employed to measure EIF2AK2 and AIM2 expression in mouse kidney tissue. Lactate dehydrogenase (LDH) activity assay was conducted to evaluate cytotoxicity. Co-immunoprecipitation (Co-IP) was performed to verify the binding relationship between EIF2AK2 and AIM2.

Results: AIM2 expression was increased in the renal tissue of mice subjected to CLP. Activation of the inflammasome and PANoptosis were observed in the renal tissue of CLP mice. AIM2 depletion attenuated PANoptosis in LPS-treated HK-2 cells. Additionally, EIF2AK2 could directly target AIM2, leading to a positive regulation of AIM2 expression. Notably, EIF2AK2 induced PANoptosis through upregulating AIM2 in HK-2 cells stimulated by LPS.

Conclusions: Our results revealed the important role of EIF2AK2-induced AIM2 upregulation in the activation of PANoptosis during septic AKI.

Keywords: AIM2; Acute kidney injury; EIF2AK2; PANoptosis; sepsis.

Figure 1.

AIM2 was highly expressed in septic AKI mice. Sepsis mouse model was established utilizing CLP surgery. (A) HE staining and PAS staining were used to detect the changes of renal tissue in mouse model. (B) Serum levels of Scr and BUN in mice were tested by kits. (C, D) AIM2 expression in renal tissue of mice was assessed with Western blot and IF. **p < .01 and ***p < .001. n = 5 mice/group.

Figure 2.

PANoptosis regulated sepsis-induced renal injury in mice. (A) Images of TUNEL staining and TUNEL-positive cells in mouse kidney tissue. (B) LDH kit was adopted to test cytotoxicity. (C) AIM2 induces PANoptosis. (D) Western blot was utilized to determine the levels of pyroptosis, apoptosis, and necroptosis-related proteins in renal tissue of mice. ***p < .001. n = 5 mice/group.

Figure 3.

AIM2 mediated sepsis-induced renal injury by regulating PANoptosis. (A) Transfection efficiency of sh-AIM2 was assessed in HK-2 cells by RT-qPCR. Next, HK-2 cells were transfected with sh-AIM2 or sh-NC, and treated with LPS. (B) Western blot detection of AIM2 expression. (C) PI staining was used to detect cell death rate. (D) LDH kit was used to test cytotoxicity. (E) Western blot was performed to determine the levels of pyroptosis, apoptosis, necroptosis and PANoptosis-related proteins. Data are presented as mean ± SD of three independent experiments. **p < .01 and ***p < .001.

Figure 4.

EIF2AK2 protein targeted and activated AIM2. (A) Western blot and IF were used to detect EIF2AK2 expression in renal tissue of Sham and CLP mice. (B) GeneMANIA database was utilized to predict the binding relationship between EIF2AK2 and AIM2. (C) Co-IP assay was used to verify the binding relationship between EIF2AK2 and AIM2. Data are the means ± SD for three independent experiments. **p < .01.

Figure 5.

EIF2AK2 targeted activation of AIM2 mediated PANoptosis in sepsis-induced renal injury. HK-2 cells were transfected with sh-EIF2AK2 or OE-AIM2, and treated with LPS. (A) Western blot detection of EIF2AK2 and AIM2 expression. (B) PI staining was used to detect cell death rate. (C) LDH kit was used to test cytotoxicity. (D) Western blot was conducted to determine the levels of pyroptosis, apoptosis, necroptosis, and PANoptosis-related proteins. Values were expressed as mean ± SD of three separate determinations. *p < .05, **p < .01, and ***p < .001.

Figure 6.

Inhibition of AIM2-mediated PANoptosis alleviated sepsis-induced renal injury in mice. The mice were subjected to different groups: Sham group, CLP group, CLP + PBS group, and CLP + A151 (AIM2 inhibitor) group. (A) HE staining and PAS staining were used to detect the changes of renal tissue in mouse model. (B) Serum levels of Scr and BUN in mice were tested by kits. (C) AIM2 expression in renal tissue of mice was assessed with Western blot. (D) Images of TUNEL staining and TUNEL-positive cells in mouse kidney tissue. (E) LDH kit was adopted to test cytotoxicity. (F) Western blot was utilized to determine the levels of pyroptosis, apoptosis and necroptosis-related proteins in renal tissue of mice. *p < .05, **p < .01, and ***p < .001. n = 5 mice/group.