Overexpression of TOLLIP Protects against Acute Kidney Injury after Paraquat Intoxication through Inhibiting NLRP3 Inflammasome Activation Modulated by Toll-Like Receptor 2/4 Signaling

Paraquat (PQ) can cause multiorgan failure including acute kidney injury (AKI). Our prior study showed that Toll-interacting protein (TOLLIP) protected against PQ-induced acute lung injury. However, the role of TOLLIP in PQ-induced AKI remains undefined. This study was aimed at understanding the role and mechanism of TOLLIP in AKI. Six-eight-week-old male Wistar rats were intraperitoneally injected with 25 mg/kg PQ to induce AKI for 24 h in vivo. HK-2 cells were treated with 300 μM PQ for 24 h to induce cellular injury in vitro or 300 μM PQ and 5 μM nuclear factor-κB (NF-κB) inhibitor BAY11-7082 for 24 h. Rats were infected with adenovirus carrying TOLLIP shRNA via tail vein injection and HK-2 cells with adenovirus carrying TOLLIP shRNA or TOLLIP 48 h before PQ exposure. Results showed that TOLLIP and Toll-like receptor 2/4 (TLR2/4) expressions were boosted in the kidney after PQ intoxication. The toxic effect of PQ on the kidney and HK-2 cells was exacerbated by TOLLIP knockdown, as evidenced by aggravated glomerulus and tubule injury, inflammatory infiltration, and cell apoptosis in the kidney and increased loss of cell viability and apoptotic cells in HK-2 cells. TOLLIP knockdown also enhanced PQ-induced NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation in vivo and in vitro and TLR2/4-NF-κB signaling in vitro, reflected by increased contents of proinflammatory cytokines and expressions of NLRP3 inflammasome-related proteins in the kidney and HK-2 cells and expressions of TLR2, TLR4, and nuclear NF-κB p65 in HK-2 cells. However, TOLLIP overexpression inhibited PQ-induced loss of cell viability, cell apoptosis, NLRP3 inflammasome activation, and TLR2/4-NF-κB signaling in vitro. Additionally, BAY11-7082 abolished TOLLIP knockdown-induced NLRP3 inflammasome activation in vitro, indicating that TOLLIP protected against NLRP3 inflammasome activation in PQ-induced AKI through inhibiting TLR2/4-NF-κB signaling. This study highlights the importance of TOLLIP in AKI after PQ intoxication.


Introduction
Paraquat (also known as methyl viologen, PQ) has been around as a widely used herbicide in agricultural production due to its ability in high-efficiency nonselective killing of leaf weeds and plants since the early 1960s [1]. However, growing evidence has pointed out its harmfulness to the mammals including human, rodents, and rabbits [2,3]. PQ intoxication is a severe threat to human health in developing countries, especially in Asia. No specific antidote has been utilized to treat PQ intoxication so far. Exposure to PQ causes severe damage in the various organ systems including the lungs, kidney, and liver [4]. The lung is the main target organ in PQ intoxication, and the respiratory failure caused by acute lung injury is the most usual cause of death in PQ intoxication patients [5]. Additionally, PQ is mainly enriched in the kidney at 3 h after exposure, which resulted in acute kidney injury (AKI) [6]. The mechanism underlying AKI after PQ intoxication, however, has not been fully understood, leading to a limitation of appropriate treatment strategy.
Toll-like receptors (TLRs), the first pattern recognition receptors, play a pivotal role in triggering innate immune responses. Activated TLRs recruit myeloid differentiation factor 88 (MyD88) via its Toll-interleukin-1 receptor (IL-1R) domain [7], and then, MyD88 binds to IL-1R-associated kinase (IRAK) via N-terminal death domain [8]. Subsequently, IRAK undergoes phosphorylation and activation, resulting in the activation of various downstream signaling pathways including nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase pathways [9]. Accumulating evidence has shown that TLRs play a key role in the pathogenesis of multiple diseases including cerebral hemorrhage [10], atherosclerosis [11], nonalcoholic fatty liver disease [12], and pulmonary fibrosis [13]. TLR-mediated signaling pathway is also involved in AKI, and inhibition of this signaling has been shown to attenuate AKI [14].
Toll-interacting protein (TOLLIP), a multifunctional protein, consists of a Tom1-binding domain in the N-terminus, a conserved 2 domain in the central region, and a coupling of ubiquitin conjugation to endoplasmic reticulum degradation domain in the C-terminus in mammals [15]. TOLLIP has been identified as a negative modulator of TLR signaling. TOLLIP interacts with TLR2 and TLR4 by binding to TLR2 and the TLR4-myeloid differentiation factor 2 complex via its C-terminus, thus suppressing TLR-mediated cellular responses by dampening the phosphorylation and activity of IRAK [16]. In addition, TOLLIP also serves as a regulator of protein sorting via its interaction with Tom1, ubiquitin, and clathrin [17]. Our prior research showed that TOLLIP was aberrantly expressed in the lung and TOLLIP overexpression exerted a protective effect on lung injury after PQ intoxication [18]. TOLLIP is also expressed in the kidney; however, whether TOLLIP is involved in AKI after PQ intoxication remains unknown.
In the present study, the expression of TOLLIP, TLR2, and TLR4 was analyzed in rats after PQ intoxication. To better understand the role of TOLLIP in AKI after PQ intoxication, the expression of TOLLIP in rats and HK-2 cells was knocked down or overexpressed and the effect of TOLLIP knockdown or overexpression on the renal or cellular injury and NLR family pyrin domain-containing 3 (NLRP3) inflammasome in vivo and in vitro was subsequently investigated. In addition, it was also verified whether the TLR2/4-NF-κB pathway was implicated in the protective mechanism of TOLLIP in PQ-induced AKI. This study provides a potential target for PQ-induced AKI treatment.

Materials and Methods
2.1. Animals. Male Wistar rats aged 6-8 weeks were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (China). All rats were housed at 22 ± 1°C under 12 h/12 h light/dark cycles and were given free access to food and water.

Animal Experimental Protocol.
In some experiments, animals were divided randomly into 2 groups (Sham and PQ groups). Rats in the PQ group were intraperitoneally injected with 25 mg/kg PQ (Aladdin, China), while rats in the Sham group were intraperitoneally injected with an equal volume of normal saline. Rats in the PQ group were euthanatized at 3, 6, 12, and 24 h after PQ injection. Rats in the Sham group were euthanatized at 24 h after saline injection. The kidney tissues were collected for further analysis.
In some experiments, animals were divided randomly into 4 groups (Sham, PQ, PQ+adenovirus-(AV-) negative control short hairpin RNA (shNC), and PQ+AV-TOLLIP short hairpin RNA (shTOLLIP) groups). Forty-eight h before PQ injection, rats in the PQ+AV-shNC and PQ+AV-shTOL-LIP groups were infected with the AV carrying shTOLLIP or shNC (10 9 PFU, tail vein injection). Rats in the PQ, PQ+AV-shNC, and PQ+AV-shTOLLIP groups were intraperitoneally injected with 25 mg/kg PQ, while rats in the Sham group were intraperitoneally injected with an equal volume of normal saline. All rats were euthanatized at 24 h after injection. The kidney tissues and serum were collected for further analysis.
All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals [19]. All procedures were reviewed and approved by the ethics committee of animal use at Shengjing Hospital of China Medical University (Accession number, 2020PS702K).

Histopathological
Analysis. The kidney tissue was embedded in paraffin. The paraffin-embedded kidney sections (5 μm) were deparaffinated in xylene and stained with hematoxylin (Solarbio) and eosin (Sangon) (H&E). Histopathological changes of the kidney tissues were observed under a microscope (OLYMPUS, Japan).

Measurement of Blood Urea Nitrogen (BUN) and Serum
Creatinine Level. The BUN and serum creatinine levels in rats were measured by their respective commercial kits (Nanjing Jiancheng, China) in accordance with the manufacturer's protocol.  , and p65 staining, tissue sections or cells were subjected to incubation with anticleaved caspase-3 (Affinity, China), anti-NLRP3 (ABclonal), and anti-p65 (Affinity) antibodies (1 : 100 dilution) at 4°C overnight, followed by incubation with Cy3-conjugated goat anti-rabbit IgG (1 : 200 dilution) at room temperature for 60 min. Next, tissue sections or cells were subjected to dihydrochloride (DAPI) (Aladdin) nuclear staining. Images were captured using a fluorescence microscope (OLYMPUS).
2.10. Cell Counting Kit-8 (CCK-8) Assay. HK-2 cells (5 × 10 3 per well) were seeded in a 96-well culture plate. After treatment, cell viability was measured using CCK-8 (KeyGEN, China). Briefly, 10 μL CCK-8 solution was added to each well and incubated for 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader (BIOTEK, USA). 2.12. Statistical Analysis. Data are expressed as mean ± SD and compared by one-way analysis of variance (ANOVA). When p < 0:05, differences were considered statistically significant. Statistical analysis was conducted by GraphPad Prism 8.0.

TOLLIP Is Upregulated in the Rat Kidney in PQ-Induced
AKI. In the rats of the PQ group, the mRNA expression levels of TOLLIP, TLR2, and TLR4 in the kidney increased gradually during PQ exposure (Figure 1(a)). In comparison with the rats of the Sham group, the mRNA expression levels of TOLLIP, TLR2, and TLR4 in the kidney were significantly increased by PQ exposure (Figure 1(a)). As shown in Figure 1(b), the rats of the Sham group showed the normal structure of the glomerulus and renal tubule, while the rats of the PQ group showed histopathological alterations in the kidney including necrosis, hemorrhage, and degenerative glomerulus and renal tubules. It has been reported that TOL-LIP interacted with TLR2 and TLR4 by binding to TLR2 and TLR4 via its C-terminus [20]. Hence, TOLLIP-TLR2 and TOLLIP-TLR4 staining was performed to verify whether the interaction between TOLLIP and TLR2/TLR4 existed in the pathogenesis of PQ-induced AKI. As shown in Figures 1(c) and 1(d), less stained area for TOLLIP and TLR2/4 in the Sham group was overlapped compared with that in the PQ group, indicating that PQ might result in more interaction between TOLLIP and TLR2/4 in the kidney tissues.

Knockdown of TOLLIP Exacerbates PQ-Induced AKI in
Rats. In order to determine whether TOLLIP was involved in PQ-induced AKI, rats were injected with the AV carrying shTOLLIP via the tail vein prior to PQ exposure. The mRNA and protein expression levels of TOLLIP in the kidney from the rats of the PQ+AV-shTOLLIP group were significantly decreased (Figures 2(a) and 2(b)). Knockdown of TOLLIP   . Staining for cleaved caspase-3 ( Figure 2(e)) showed that PQ exposure increased cleaved caspase-3 expression in the rat kidney but knockdown of TOLLIP further increased cleaved caspase-3 expression in the kidney. These data suggested that TOLLIP played a protective role in PQ-induced AKI.

Knockdown of TOLLIP Promoted the PQ-Induced
Activation of NLRP3 Inflammasome in the Rat Kidney. Subsequently, the effect of TOLLIP knockdown on the activation of NLRP3 inflammasome in the rat kidney was investigated. As shown in Figure 3( (Figure 3(b)) also exhibited further increased expression levels of NLRP3, ASC, and caspase-1 in the kidney from the rats of the PQ+AV-shTOLLIP group. Consistent with Western immunoblot analysis, staining for NLRP3 (Figure 3(c)) showed that knockdown of TOLLIP further increased the NLRP3 expression induced by PQ exposure in the kidney. These data indicated that TOLLIP inhibited the PQ-induced activation of NLRP3 inflammasome in the rat kidney.

Discussion
PQ intoxication leads to a large amount of deaths, whose mortality rate is as high as 60%~80% [21]. The kidney is also a major target organ after PQ intoxication apart from the    Mediators of Inflammation lung. Therefore, it is necessary to explore the mechanism underlying AKI after PQ intoxication. It is widely accepted that TOLLIP is a negative regulator of inflammatory response. In our prior study, the expression of TOLLIP was downregulated in the lung in rats after PQ intoxication and TOLLIP overexpression mitigated PQinduced acute lung injury [18]. In contrast to our prior study, the present study showed that the expression of TOL-LIP was upregulated in the kidney after PQ intoxication. It has been reported that TOLLIP was increased in lung injury after cecal ligation and puncture [22] but decreased in hepatic ischemia-reperfusion injury [23]. Therefore, TOL-LIP may exert different roles in the pathogenesis of different disorders. Subsequently, the effect of TOLLIP inhibition on AKI after PQ intoxication was analyzed in the present study. As a result, TOLLIP inhibition obviously aggravated kidney injury as evidenced by renal pathological changes using H&E staining. BUN and serum creatinine are commonly determined to evaluate renal function [24]. Increased levels of BUN and creatinine were found in the serum of PQpoisoning patients [25]. The present study showed that PQ-induced increases in BUN and serum creatinine level were promoted by TOLLIP inhibition. These findings reflected loss of renal function by TOLLIP inhibition. Similar with our prior study, TOLLIP also showed a protective role in PQ-induced kidney injury.

Mediators of Inflammation
PQ has been reported to trigger kidney cell apoptosis in vivo and in vitro [26,27]. TOLLIP functions as a positive or negative regulator of cell apoptosis. TOLLIP has been demonstrated to inhibit bleomycin-induced bronchial epithelial cell apoptosis and IFN-γ-and TNF-α-induced intestinal epithelial cell apoptosis [28,29]. However, TOLLIP could promote apoptosis of cardiomyocytes and hepatocytes [23,30]. In the present study, TOLLIP inhibition increased the expression of cleaved caspase-3 in the kidney tissues and HK-2 cells and cleaved PARP expression in HK-2 cells as well as the percentage of apoptotic HK-2 cells, indicating increased PQ-induced kidney cell apoptosis by TOLLIP inhibition. In in vitro experiments, TOLLIP upregulation led to the opposite results. These finding indicated that TOLLIP protected kidney cells from PQ-induced apoptosis.
Aberrant activation of NLRP3 inflammasome is closely related to the development of various diseases, including inflammatory diseases [31], infections [32], Alzheimer's disease [33], and diabetes [34]. The NLRP3 inflammasome comprises the sensor NLRP3, the adaptor ASC, and the effector serine caspase-1 [35]. Multiple bacterial products or danger signals have been shown to induce activation of NLRP3 inflammasome [36]. Emerging evidence has elucidated that its activation was a contributor of renal inflammation, fibrosis, and ischemia-induced AKI [37,38]. In recent years, excessive activation of NLRP3 inflammasome was found in the kidney after PQ intoxication [25]. Activation of NLRP3 inflammasome induces caspase-1 activation, which results in cleavage of pro-IL-1β and pro-IL-18 into their active forms IL-1β and IL-18 [39]. Consistent with a previous study, the present study showed that PQ increased the expression of NLRP3, ASC, and caspase-1 and secretion of IL-18, IL-1β, IL-6, and TNF-α in the kidney or HK-2 cells. Significantly, TOLLIP inhibition promoted the PQ-induced increases in vivo and in vitro. However, TOLLIP upregulation abrogated the PQ-induced increases in vitro. The findings first revealed the role of TOLLIP in activation of NLRP3 inflammasome in AKI. As reported, NLRP3 inflammasome induced cell apoptosis during kidney injury [40,41]. Hence, TOLLIP-induced reduction in cell apoptosis during PQ intoxication might be attributed to blockage of NLRP3 inflammasome activation.
TOLLIP has been generally considered as a negative regulator of the TLR-mediated signaling pathway [42]. Activation of the TLR-mediated signaling pathway has been involved in the pathogenesis of multiple renal diseases, including fibrosis [43], ischemia/reperfusion injury [44], glomerulonephritis [45], and AKI [46]. Blockade of the TLRmediated signaling pathway via MyD88 has been reported to mitigate lung injury after PQ intoxication [47]. In this present study, PQ enhanced the expression of TLR2 and TLR4 in the rat kidney. TOLLIP inhibition promoted PQinduced expression of TLR2, TLR4, and nuclear p65 and p65 nuclear translocation in vitro, while TOLLIP upregulation eliminated the PQ-induced activation of TLR2/4 and its downstream NF-κB signaling. The TLR2/4-mediated signaling pathway triggers the activation of NLRP3 inflammasome [48]. To confirm whether TLR2/4-mediated signaling was involved in TOLLIP-induced inhibition in NLRP3 inflammasome activation, NF-κB inhibitor was utilized to treat HK-2 cells after TOLLIP knockdown. The NF-κB inhibitor was found to reverse the activation of NLRP3 inflammasome induced by TOLLIP knockdown, indicating that the function of TOLLIP in NLRP3 inflammasome was at least partially mediated by TLR2/4-NF-κB signaling. Besides, mitogen-activated protein kinase (MAPK) pathway was also a downstream signaling pathway of TLR signaling. Likewise, the MAPK signaling pathway induces the activation of

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Mediators of Inflammation NLRP3 inflammasome in kidney injury [49]. Therefore, it can be investigated in the future whether the MAPK pathway is implicated in PQ intoxication-caused activation of NLRP3 inflammasome and inflammatory cascades.

Conclusion
In summary, TOLLIP and TLR2/4 expressions were boosted in the kidney after PQ intoxication. TOLLIP inhibition exacerbated PQ-induced kidney and cellular injury and activation of NLRP3 inflammasome in rats and HK-2 cells and enhanced TLR2/4 and its downstream NF-κB signaling pathway in HK-2 cells. However, TOLLIP upregulation had an opposite effect on PQ-induced cellular injury and activation of NLRP3 inflammasome as well as TLR2/4 and its downstream NF-κB signaling pathway in HK-2 cells. In addition, the NF-κB inhibitor restored the activation of NLRP3 inflammasome induced by TOLLIP inhibition upon PQ exposure in HK-2 cells. These data indicated that the protective role of TOLLIP against NLRP3 inflammasome activation in PQinduced AKI was at least partially mediated by TLR2/4-NF-κB signaling. This study highlights the importance of TOL-LIP in AKI after PQ intoxication.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.