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

Abstract

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.

Figure 1

Effect of PQ on the expression of TOLLIP, TLR2, and TLR4 in the rat kidney tissues. Wistar rats received 25 mg/kg PQ via intraperitoneal injection. (a) The mRNA expression of TOLLIP, TLR2, and TLR4 in the kidney tissues at 3, 6, 12, and 24 h post-PQ treatment. (b) Representative images of H&E staining of the kidney tissues at 24 h post-PQ treatment (red asterisk, degenerative renal tubules; black arrow, necrosis; yellow arrow, degenerative glomerulus; and green double arrowhead, hemorrhage). (c, d) Representative images of TLR2-TOLLIP and TLR4-TOLLIP staining of the kidney tissues at 24 h post-PQ treatment. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (n = 8).

Figure 2

Effect of TOLLIP knockdown on the PQ-induced AKI in rats. Wistar rats were infected with the AV carrying shTOLLIP via tail vein injection and then treated with 25 mg/kg PQ via intraperitoneal injection 48 h post-AV infection. (a, b) The mRNA and protein expression of TOLLIP in the kidney tissues at 24 h post-PQ treatment. (c) The level of BUN and creatinine in the serum at 24 h post-PQ treatment. (d) Representative images of H&E staining of the kidney tissues at 24 h post-PQ treatment. (e) Representative images of cleaved caspase-3 immunofluorescence staining of the kidney tissues at 24 h post-PQ treatment. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (n = 8).

Figure 3

Effect of TOLLIP knockdown on the PQ-induced production of NLRP3 inflammasome in the rat kidney tissues. Wistar rats were infected with the AV carrying shTOLLIP via tail vein injection and then treated with 25 mg/kg PQ via intraperitoneal injection 48 h post-AV infection. (a) The level of IL-18, IL-1β, TNF-α, and IL-6 in the kidney tissues at 24 h post-PQ treatment. (b) The protein expression of NLRP3, ASC, pro-caspase-1, and caspase-1 in the kidney tissues at 24 h post-PQ treatment. (c) Representative images of NLRP3 immunofluorescence staining of the kidney tissues at 24 h post-PQ treatment. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (n = 8).

Figure 4

Effect of TOLLIP knockdown and overexpression on the PQ-induced loss of cell viability and apoptosis of HK-2 cells. HK-2 cells were infected with the AV carrying shTOLLIP or TOLLIP and then treated with 300 μM PQ 48 h post-AV infection. (a) Cell viability was measured by CCK-8 assay at 24 h post-PQ treatment. (b, c) Cell apoptosis was determined by Annexin V-FITC-PI staining at 24 h post-PQ treatment. (d) The protein expression of TOLLIP, cleaved caspase-3, and cleaved PARP at 24 h post-PQ treatment. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (n = 3).

Figure 5

Effect of TOLLIP knockdown and overexpression on the PQ-induced activation of NLRP3 inflammasome in HK-2 cells. HK-2 cells were infected with the AV carrying shTOLLIP or TOLLIP and then treated with 300 μM PQ 48 h post-AV infection. (a) The protein expression of TLR2, TLR4, NLRP3, pro-caspase-1, caspase-1, and nuclear p65 at 24 h post-PQ treatment. (b) The level of IL-18 and IL-1β at 24 h post-PQ treatment. (c, d) Representative images of p65 and NLRP3 immunofluorescence staining at 24 h post-PQ treatment. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (n = 3).

Figure 6

Role of the NF-κB pathway in the TOLLIP-induced inhibition of NLRP3 inflammasome activation in PQ-treated HK-2 cells. HK-2 cells were infected with the AV carrying shTOLLIP and then treated with 300 μM PQ and 5 μM BAY11-7082 48 h post-AV infection. (a) The level of IL-18 and IL-1β at 24 h post-PQ and BAY11-7082 treatment. (b) The protein expression of NLRP3, pro-caspase-1, and caspase-1 at 24 h post-PQ and BAY11-7082 treatment. ^∗p < 0.05 and ^∗∗p < 0.01 (n = 3).