AIM2 Inflammasome Contributes to Aldosterone-induced Renal Injury via Endoplasmic Reticulum Stress

Inflammatory response and renal fibrosis are the hallmarks of chronic kidney disease (CKD). However, the specific mechanism of aldosterone-induced renal injury in the progress of CKD requires elucidation. Emerging evidence has demonstrated that absent in melanoma 2 (AIM2)mediated inflammasome activation and endoplasmic reticulum stress (ERS) play a pivotal role in the renal fibrosis. Here, we investigated whether overexpression or deficiency of AIM2 affects ERS and fibrosis in aldosterone-infused renal injury. Interestingly, we found that AIM2 was markedly expressed in the diseased proximal tubules from human and experimental chronic kidney disease. Mechanically, overactivation of AIM2 aggravated aldosterone-induced ERS and fibrotic changes in vitro while knockdown of AIM2 blunted these effects in vivo and vitro. By contrast, AIM2 deficiency ameliorated renal structure and function deterioration, decreased proteinuria levels and lower systolic blood pressure in vivo; silencing of AIM2 blocked inflammasome-mediated signaling pathway, relieved ERS and fibrotic changes in vivo. Furthermore, mineralocorticoid receptor antagonist eplerenone and ERS inhibitor tauroursodeoxycholic acid (TUDCA) had nephroprotective effects on the basis of AIM2 overactivation in vitro while they failed to produce a more remarkable reno-protective effect on the treatment of AIM2 silence in vitro. Notably, the combination of TUDCA with AIM2 knockdown significantly reduced proteinuria levels in vivo. Additionally, immunofluorescence assay identified that apoptosis-associated speck-like protein (ASC) recruitment and GasderminD (GSDMD) cleavage respectively occurred in the glomeruli and tubules in vivo. These findings establish a crucial role for AIM2 inflammasome in aldosterone-induced renal injury, which may provide a novel therapeutic target for the pathogenesis of CKD. D ow naded rom http://pndpress.com /clinsci/ardf/doi/10.1042/C S202110754/cs-2021-1075.pdf by gest on 24 D ecem er 2021 C liical Scnce. This is an Acepted M ancript. ou re encuraged to se he Vrsion of R eord tat, w en puished, w ill relace his vesion. he m st up-tote-version is avilable at https://rg/10.1042/C S211075


Introduction
Chronic kidney disease (CKD) is characterized by the irreversible damage of renal function and renal fibrosis, but the pathogenetic mechanisms of CKD still have not been completely elucidated [1]. As the major component in the renin-angiotensin-aldosterone system (RAAS), aldosterone activates the mineralocorticoid receptor (MR) and plays a critical role in the pathogenesis of chronic kidney disease [2,3]. It not only regulates salt and water homestasis, but also induces glomerulosclerosis and tubular epithelial cells apotosis, and even aggravates the progression of end-stage renal disease (ESRD) [4][5][6][7].
Absent in melanoma 2 (AIM2), as one of the major members in the absent in melanoma-2like receptor (ALRs) family, is a novel inflammatory factor of interferon (IFN)-inducible multiprotein complex located in the cytoplasm [8]. In general, AIM2 contains a N-terminal PYD and C-terminal HIN domains, and the interaction between the PYD and HIN domains causes AIM2 molecule to be in a self-inhibiting state [9]. However, in response to "dangerous signal" or "injury signal", AIM2 senses and binds cytoplasmic double stranded DNA (dsDNA) from necrotic cells, then recruits Caspase 1 and apoptosis-associated speck-like protein (ASC) to reach the assembly of polycomplex inflammasome [10]. Subsequently, AIM2 inflammasome activation would trigger the maturation and release of proinflammatory cytokine, such as IL-1 β and IL-18. Furthermore, it also fosters the cell membrane perforation to achieve the cleavage of Gasdermin-D (GSDMD), eventually resulting in cells pyroptosis [11,12].
Emerging research progress has indicated that AIM2 inflammasome is strongly linked to the pathogenesis of renal diseases. To date, serval studies have been proved that AIM2 expression is closely associated with the pathological grade of lupus nephritis [13,14].
Moreover, increased AIM2 expression was related to the high titer level of anti-dsDNA in plasma of systemic lupus erythematosus (SLE) mice, and knockdown of AIM2 also significantly inhibited macrophage activation and inflammation, thus relieving the symptoms of SLE [15]. More strikingly, Komada and colleagues reported that AIM2 inflammasome aggravated renal fibrosis and triggered inflammation in unilateral ureteral obstruction (UUO) mice model through macrophage uptake of necrotic cells dsDNA [16]. Nevertheless, increasing Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20211075/927034/cs-2021-1075.pdf by guest on 24 December 2021 literature demonstrates that inflammasome activation could trigger reactive oxygen species (ROS) synthesis, endoplasmic reticulum stress and mitochondrial dysfunction in the course of innate immune response [17]. Previously, we reported that NLRP3 inflammasome mediated mitochondrial ROS, leading to aldosterone-infused renal injury, and knockout NLRP3 gene significantly attenuates aldosterone-induced renal fibrosis [18]. Endoplasmic reticulum stress (ERS), which is featured with misfolded proteins accumulation and disorder in the endoplasmic reticulum in response to pathological injury signal, plays an important role in the progression of chronic kidney disease [19]. Also, our previous findings demonstrated that aldosterone contributed to podocytes injury and tubular epithelial cells apoptosis via ERS both in vivo and in vitro [5,20,21]. As a distinctive inflammatory mediator, AIM2 inflammasome has not been extensively studied in chronic kidney disease. However, it remains elusive whether and how AIM2 inflammasome mediates aldosterone-infused renal injury via ERS. In addition, the effects of combining AIM2 inflammasome overactivation or knockdown with MR antagonist eplerenone or ERS inhibitor TUDCA on the aldosterone-infused injury model still remains unclear.
The current study intends to explore the role of AIM2 inflammasome and ERS in aldosterone-induced renal injury and its related mechanism, and we are supposed to provide the new perspective and target in the progression and treatment of chronic kidney disease.

Human renal biopsy samples
Excessive aldosterone induces the hyperactivation of MR, which exacerbates glomerulosclerosis and renal interstitial fibrosis, and aggravates the progression of chronic kidney disease [22,19]. The animal aldosterone-infused injury models were also established to induce renal fibrotic changes [2,7]. Accordingly, to investigate the potential role of AIM2 in renal fibrosis, we screened and collected human renal biopsy specimens of CKD patients with Stage G1-G4 (n=12) and normal nephrectomy samples adjacent to renal carcinoma (n=8) at All participants have accepted and signed the informed consent documents before their enrollment.

Biochemical Measurements
The serum creatinine, blood urea nitrogen and 24-hour urine protein quantitation levels of mice were measured by an automatic chemistry analyzer (Roche c702, KingMed Diagnostics, Shanghai, China).

Western blotting
The total protein from kidney issues and cells were lysed with RIPA buffer (

RNA extraction and real-time PCR
The total mRNA from kidneys and cells were extracted by using Trizol Reagent (Thermo  (Table S1). The mouse kidneys were fixed in 4% paraformaldehyde and made into paraffin, and then were cut at 3-μm thickness. The glomerulus sclerosis was estimated by periodic acid-schiff (PAS) staining, which was indicated based on the percentage of glomerular sclerosis area to the total area. The glomerulus sclerosis score was categorized as the following 5 grades: 0; 1,10%; 2, 10%-25%; 3, 26%-50%; score 4, 51%-75%; score 5, >75%.

Cytokine measurement by ELISA
The serum IL-18 levels were quantified by the mouse IL-18 ELISA kit (70-EK218-96, Lianke biotech, Hangzhou, China). A standard curve was drawn according to the manufacturer's protocol. Each value was expressed as pg/ml.

Statistical analysis
The data are shown as the mean ±SEM and were analyzed by SPSS version 23.0 (IBM Corp.,).
Column graphs were drawn by using GraphPad Prism version 8.0 (San Diego, CA).
Comparisons between groups were conducted by the Student t-test (2 groups) and one-way analysis of variance with post hoc analysis using Tukey's test as appropriate. P < 0.05 was considered statistically significant.

AIM2 is upregulated predominantly in the renal biopsy of CKD patients.
To determine the involvement of AIM2 in the pathogenesis of CKD-induced renal fibrosis, we first examined AIM2 distribution and expression in human kidney issues from normal healthy control group and CKD patients. We screened the patients with CKD Stage G1-G4 whose renal biopsies were pathologically diagnosed as IgA nephropathy with glomerulosclerosis index more than 50% according to the KIDGO guidelines. A total of 12 CKD patients were enrolled in this study, and normal nephrectomy samples adjacent to renal carcinoma (n=8) served as normal control group. All patients' clinical and laboratory data were also displayed in the Table   1. As shown in the Figure 1, the contents of MR protein were significantly increased in the kidney of CKD patients. Compared with normal control group, both renal cortex and medulla staining expression of AIM2 was upregulated predominantly among CKD group (Figure 2a-d).
Then, we used double immunofluorescence staining with proximal tubule biomarker Megalin to detect the specific cellular location of AIM2 in the human kidney issues. Strikingly, AIM2 was mainly found in the diseased proximal tubules area in CKD patients compared with normal control group ( Figure 3). Together, these results provide the novel clinical evidence that AIM2 is closely correlated to aldosterone-induced chronic kidney disease. however, AIM2 knockdown and TUDCA attenuated these effects (Figure 4i, j). Taken together, these findings illustrate that knockdown of AIM2 ameliorates the damage of renal pathology and renal function, reduces proteinuria levels and lowers systolic blood pressure in aldosteroneinfused injury mice.

AIM2 knockdown blocks inflammasome-mediated signaling pathway, relieves ERS and fibrotic changes in aldosterone-induced injury mice model.
We next investigated the mechanism whereby AIM2 knockdown in the aldosterone-induced renal fibrosis in vivo. As shown in the Figure 5a

AIM2 is concentration-dependently and time-dependently induced by aldosterone in cultured both glomerular mesangial cells and tubular epithelial cells.
To explore the specific role of AIM2 in the aldosterone-induced glomeruli and tubules injury, our findings indicated that the expression of AIM2 protein was markedly induced by the different concentrations of aldosterone in SV40MES13 and mTEC (Figure 7a-d). Subsequently, we respectively selected the optimal concentration of aldosterone to simulate SV40MES13 and mTEC within different time. Similarly, AIM2 induction was significantly upregulated by aldosterone with the increase of time (Figure 7a-d). These results demonstrate that aldosterone induces AIM2 protein expression in a concentration-dependent and time-dependent manner.

Overactivation of AIM2 aggravates aldosterone-induced ERS and fibrosis changes in both glomerular mesangial cells and tubular epithelial cells.
To investigate the effects of AIM2 overactivation on both glomerular mesangial cells and tubular epithelial cells, we transfected poly(dA: dT) to achieve the overexpression of AIM2 inflammasome. The immunoblotting results showed that the combination of both aldosterone and poly(dA: dT) markedly activated AIM2, cleaved-Caspase1, IL-1βas well as ERS marker GRP78, and also significantly increased fibrosis marker protein expression, includingα-SMA and collagen I (Figure 8a-d). These findings indicate that overactivation of AIM2 further triggers the release of inflammatory mediators and induced ERS, which aggravates aldosteroneinduced renal fibrosis.

Deficiency of AIM2 attenuates aldosterone-induced ERS and fibrosis changes in both glomerular mesangial cells and tubular epithelial cells.
To examine the critical effects of AIM2 knockdown, we next tested whether silencing of AIM2 by shRNA affects aldosterone-induced ERS and renal fibrosis level. The immunoblotting findings showed that sh-AIM2 remarkably decreased the protein expression of cleaved-Capase-1, IL-1β, GRP78, α-SMA and collagen I (Figure 9a-h). These results suggest that AIM2 deficiency suppresses aldosterone-induced inflammation and ERS, as well as attenuating renal fibrosis.

MR antagonist eplerenone and ERS inhibitor TUDCA inhibits aldosterone-induced ERS and inflammation in both glomerular mesangial cells and tubular epithelial cells.
To investigate the further insights of nephroprotective mechanism, both SV40MES13 and mTEC were treated additively with MR antagonist eplerenone and ERS inhibitor TUDCA in the presence of AIM2 overactivation. As expected, eplerenone and TUDCA significantly reduced the protein levels of AIM2, cleaved-Caspase 1, IL-1β, GRP78 ,α-SMA and Collagen I (Figure 8a-d). Furthermore, to identify the combined reno-protective impacts, both protective effects on aldosterone-induced renal injury (Figure 9c, d, g, h). These data indicate that eplerenone and TUDCA alleviate aldosterone-induced ERS and inhibit inflammation caused by the excessive activation of AIM2 inflammasome while they fail to cooperatively have a more protective effect on the basis of AIM2 gene silencing.

Discussion
Persistent inflammation and renal fibrosis are the prominent feature and common pathway leading to chronic kidney disease. As the indispensable mineralocorticoid hormone, aldosterone acts on various renal cells and vessels, including promoting epithelial mesenchymal transformation, increasing extracellular matrix and collagen deposition, and aggravating vascular remodeling, which incurs issue inflammation injury, and eventually exacerbates glomerulosclerosis and tubulointerstitial fibrosis [7]. AIM2 inflammasome, the essential mediator of innate immune response, recognizes pathogen-associated molecular patterns or damage-associated molecular patterns, and triggers the maturation and release of proinflammatory cytokines, such as IL-1β, IL-18 and GSDMD, thus inducing inflammation and even pyroptosis [23]. Notably, we identified that AIM2 inflammasome, a multiprotein complex with PYD and HIN domains, played a crucial role in the pathogenesis of chronic kidney disease. In this study, we provide the new evidence that AIM2 inflammasome activation mediates aldosterone-induced ERS and exacerbates renal fibrosis in vitro and in vivo.
It has been reported that AIM2 was distributed constitutively in glomerulus and to a lesser extent in tubular epithelial cells in normal healthy human group; however, the protein expression of AIM2 was significantly upregulated in tubular epithelium and in infiltrating leukocytes in the renal biopsy of CKD patients diagnosed as hypertensive nephropathy and diabetic nephropathy [16]. Strikingly, we also found that AIM2 was markedly increased among nephritis [15,27]. Notably, Chai and colleagues found that overactivation of AIM2 promotes hepatocellular carcinoma metastasis to inhibit the invasion of renal carcinoma [28], and they subsequently designed H1/pAIM2 nanoparticles to provide a novel therapeutic target for renal carcinoma [29,30]. Recently, they further found that AIM2 exerted significant anti-tumour effects in treatment of renal cancer [31], whereas M1 macrophages activated by AIM2 would foster the rejection of renal carcinoma [32]. Hence, these results provide adequate evidence for the immunological mechanism of AIM2 involvement in renal diseases or cancers. Thus, we cannot rule out the significance that aldosterone mediates AIM2 inflammasome activation in other renal immune cells or inflammatory cells, such as T cells, B cells or resident macrophages in the kidney. Indeed, it is essential to acknowledge the role of AIM2 inflammasome in other different types of renal cells to get a comprehensive understanding of aldosterone-induced injury mechanism.
AIM2 inflammasome-mediated signals are mainly categorized into two approaches: canonical and non-canonical functions [33]. Initially, AIM2 is composed of PYI and HIN domains, senses pathogen signals or host-derived dsDNA, and then recruits Caspase 1 to form the canonical inflammasome complex requiring ASC specks as a binding bridge [11,34,35].
Interestingly, we observed that ASC specks were significantly localized in the glomerulus of aldosterone-infused mice model while GSDMD was predominantly expressed in the renal tubule though immunofluorescence assay. In our opinion, we speculated that their distribution and expression discrepancy may be due to AIM2 inflammasome-mediated different inflammation signaling pathways in the mouse glomeruli and tubules. deficiency attenuates apoptosis through the activation of mitochondrial oxidation and autophagy in contrast-induced mouse AKI model [37]. Our previous researches also have indicated that knockout of NLRP3 gene alleviated mitochondrial impairment and reversed renal fibrosis in mouse UUO model of CKD [18]. In fact, Komada and colleagues have identified that no matter single geen deficiency (AIM2 -/-or NLRP3 -/-) or double geen deficiency (AIM2-/-NLRP3-/-) has remarkable protective effects on the UUO-induced renal fibrosis. Of note, the double deficient mice (AIM2-/-NLRP3-/-) failed to confer a more significant nephroprotective benefit than single AIM2 deficient (AIM2-/-) and single NLRP3 deficient (NLRP3-/-) mice [16].
These findings suggest that both AIM2 and NLRP3 inflammasome play an indispensable role in the renal inflammation and fibrosis injury, whereas the protective effect of double AIM2 and NLRP3 knockout is approximately equivalent to single AIM2 or NLRP3 deficiency. Since the AIM2 or NLRP3 inflammasome activation-mediated innate immunology also matters in response to injury signal, both AIM2 and NLRP3 deficiency may alleviate the inflammatory response, but it might cause a lack of innate immunity, which may explain why it fails to generate a more significant renal protective effect. Therefore, further investigations are needed to elucidate the specific mechanism of discrepancy between AIM2 and NLRP3 inflammasome in aldosterone-induced renal fibrosis.
Eplerenone is the classical MR antagonist, which has been widely applied into clinical treatment of CKD and aldosterone-infused injury animal models. In addition to significant antiinflammatory and anti-fibrotic effects, we also observed that eplerenone attenuated aldosterone-induced ERS on the basis of AIM2 overactivation in vitro while it failed to produce a superimposed nephroprotective effect when combined with AIM2 silence. Mounting evidence has suggested that ERS plays an essential role in the pathogenesis of CKD, and aggravates the progression of ESRD [38,20,39,40]. Our previous findings have identified that ERS inhibitor TUDCA is able to relieve inflammatory response and renal fibrosis in the aldosterone-infused mouse model [41], and also have significant cardioprotective effects on the uremic rat model [42]. For the sake of investigating the combined reno-protection, we applicated TUDCA on the basis of AIM2 knockdown in aldosterone-infused injury mice. However, except for the was also the upstream of ERS. Indeed, the distinctive interplay between ERS and different inflammasome activation in renal diseases has been not completely elucidated. Based on these data, ongoing researches are required to investigate the distinct cross-talk between ERS and AIM2 inflammasome.
In conclusion, our findings demonstrate that AIM2 inflammasome mediates inflammation activation and ERS in the process of aldosterone-induced renal injury. Overexpression of AIM2 significantly aggravates aldosterone-infused ERS and fibrotic changes in vitro; by contrast, silencing of AIM2 effectively ameliorates aldosterone-induced ERS and renal fibrosis in vivo and in vitro. Hence, AIM2 inflammasome may provide a novel therapeutic target to prevent chronic kidney disease.

Clinical perspectives
(i) Chronic kidney disease (CKD) is characterized by persistent inflammation and renal fibrosis; AIM2 inflammasome activation and endoplasmic reticulum stress (ERS) play a pivotal role in the renal diseases. However, the specific mechanism of aldosterone-induced renal injury in the progress of CKD needs elucidation.
(ii) AIM2 markedly increases in the renal biopsy of CKD patients. AIM2 inflammasome

Data availability
Data that support the findings of this study are available upon request from the corresponding author.

Conflicts of interest
All authors declare no conflicts of interest.

Funding
This research was financially supported by the National Natural Science Foundation of China

Ethic statement
The present study was approved by the Ethics Committee on Human Research of Huashan Hospital, Fudan University. All participants have accepted and signed the informed consent documents before their enrollment. Supplementary material Table S1. The mouse gene primer pairs and sequences for RT-PCR are listed. Figure S1. The relative mRNA levels of ASC and CHOP in the kidney issue of aldosteroneinfused injury mice model.        . All values were presented as mean±SEM (n=6). ***p < 0.001 compared with normal control group.

Figure 2 AIM2 markedly increases in the renal biopsy of patients with CKD.
Representative micrographs of AIM2 expression in the renal cortex (a) and medulla (c) area among both normal heathy group (n=8) and CKD patients (n=12). Quantitative determination of AIM2 expression in both renal cortex (b) and medulla (d). ×400, bar=50μm. All values were presented as mean±SEM (n=6). ***p < 0.001 compared with normal healthy group.