NLRP3 inflammasome in rosmarinic acid-afforded attenuation of acute kidney injury in mice

Abstract

Cisplatin (CP) is a well-known anticancer drug used to effectively treat various kinds of solid tumors. CP causes acute kidney injury (AKI) and unfortunately, there is no therapeutic approach in hand to prevent AKI. Several signaling pathways are responsible for inducing AKI which leads to inflammation in proximal convoluted tubule cells in the kidney. Furthermore, the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome is involved in the CP-induced AKI. In this study, we investigated therapeutic effects of rosmarinic acid (RA) against inflammation-induced AKI. RA was orally administered at the dose of 100 mg/kg for two consecutive days after 24 h of a single injection of CP at the dose of 20 mg/kg administered intraperitoneally in Swiss albino male mice. Treatment of RA inhibited the activation of NLRP3 signaling pathway by blocking the activated caspase-1 and downstream signal molecules such as IL-1β and IL18. CP activated HMGB1-TLR4/MyD88 axis was also found to be downregulated with the RA treatment. Activation of nuclear factor-κB and elevated protein expression of cyclooxygenase-2 (COX-2) were also found to be downregulated in RA-treated animals. Alteration of early tubular injury biomarker, kidney injury molecule-1 (KIM-1), was found to be subsided in RA-treated mice. RA has been earlier reported for antioxidant and anti-inflammatory properties. Our findings show that blocking a critical step of inflammasome signaling pathway by RA treatment can be a novel and beneficial approach to prevent the CP-induced AKI.

Figure 1

Effect of RA on kidney histological morphology changes in various groups of animals. In (a), kidney section of control group animal treated with normal saline shows normal histoarchitecture with smooth and homogenous cellular structure. Normal glomeruli are represented by a black arrow. In (b), kidney section of CP-treated mouse shows altered histology with necrotic changes, degenerated glomeruli and tubular damage represented by the black arrows. In (c), kidney section of mouse treated with RA along with CP depicts a significant restoration in histology. However, some degenerated glomeruli still persisted with intact and normal glomeruli. (d) RA alone treatment showed no adverse changes in histology of mouse kidney section. In (e), histogram represents significant changes after CP and RA administration in mouse kidney sections and (f) histogram represents the inflammation scores. Treatment of CP caused a significant increase in histological changes (**p < 0.01) and significant infiltration of inflammatory cells (***p < 0.001) when compared with the control group of animals. Treatment of RA attenuated the CP-induced tubular injury and inflammation in a significant manner (^#p < 0.05) and (^##p < 0.01), respectively in CP + RA group when compared with the CP-treated group. Scale bars: 50 μm; original magnification: × 40.

Figure 2

Immunohistochemical analysis of NLRP3, caspase-1, IL-1β and KIM-1 expression after the administration of CP and RA. In (a), dark brown staining represents an increase in the expressions of NLRP3, caspase-1, IL-1β and KIM-1 protein as a result of adverse effects of CP in kidney tissue when compared with control group of animals. On the other hand, faint brown color indicates positive anti-inflammatory effect of RA against CP toxicity in CP + RA100 treated group when compared with CP alone group. There was no remarkable change in RA alone treated group of animals when compared with control group of animals. Histograms in (b–e) represent significant differences in the expression of NLRP3, caspase-1, IL-1β and KIM-1 proteins, respectively after CP and RA treatments. The administration of CP caused a significant increase in the expressions of NLRP3 (***p < 0.001) (b), caspase-1 (***p < 0.001) (c), IL-1β (***p < 0.001) (d) and KIM-1 (***p < 0.001) (e) when compared with control group of animals. However, treatment of RA along with CP showed its protection via attenuating these changes significantly in NLRP3 (^#p < 0.05), caspase-1 (^#p < 0.05), IL-1β (^##p < 0.01) and KIM-1 (^#p < 0.05) when compared with the CP-treated animals. No significant change was observed in RA only treated animals. Interquartile ranges are shown by a box and the median value is indicated by a line across the box. Statistical analysis was performed using Kruskal–Wallis tests (ANOVA on Ranks). Scale bars: 50 μm, original magnification: × 40.

Figure 3

mRNA expression analysis of NLRP3, caspase-1, IL-1β, IL-18 and IL-6 in the kidney tissue after the administration of CP and RA. The treatment of CP at the dose of 20 mg/kg caused a significant upregulation in the expression of NLRP3 (**p < 0.01) (a), caspase-1 (***p < 0.001) (b), IL-1β (***p < 0.001) (c), IL-18 (**p < 0.01) (d) and IL-6 (***p < 0.001) (e) at mRNA levels when compared with control group of animals. On the other hand, the administration of RA at the dose of 100 mg/kg caused a significant down-regulation in the expression of NLRP3 (^#p < 0.05), caspase-1 (^##p < 0.01), IL-1β (^#p < 0.05), IL-18 (^#p < 0.05) and IL-6 (^#p < 0.05) at mRNA level when compared with CP-treated group of animals. There was no significant difference in RA only treated animals when compared with control group of animals. Each value is represented as mean ± SEM (n = 6).

Figure 4

Western blot analysis and densitometric quantification of hallmarks of NLRP3 inflammasome activation, NLRP3 and ASC proteins in the mouse kidney treated with CP and RA. (a) Represents the immunoblots of NLRP3 and ASC proteins in CP and RA treatments in mouse kidney. The administration of CP at the dose of 20 mg/kg b.wt. caused an increase in the expression of NLRP3 and ASC proteins when compared with control group of animals. While the treatment with the dose of 100 mg/kg b.wt. of RA along with CP caused a decrease in the expression of NLRP3 and ASC proteins when compared with CP alone treated group of mice. In this figure histogram (b) and (c) represent significant differences between CP and CP + RA100 treated group of animals. The treatment of CP caused a significant increase in the expression of NLRP3 (**p < 0.01) and ASC (***p < 0.001) proteins when compared with control group of animals. However, the treatment of RA along with CP caused a significant restoration in the expression of NLRP3 (^#p < 0.05) and ASC (^#p < 0.05) proteins when compared with CP alone treated group of mice. No significant differences were noticed in RA alone treated group of animals when compared with the control group of animals. Data are expressed as means ± SEM (n = 6).

Figure 5

Analysis of activated caspase-1 by western blotting after CP and RA administration in mouse kidney. (a) Represents the immunoblots of activated cleaved subunits (p10 caspase-1 and p20 caspase-1) of caspase-1 after CP and RA treatments. The administration of CP caused an increase (dark bands) in the expression of cleaved p10 caspase-1 and p20 caspase-1 proteins when compared with control group of animals. While the treatment of RA along with the administration of CP caused a decrease (light bands) in the expression of p10 caspase-1 and p20 caspase-1 proteins when compared with the CP alone treated group of mice. Histogram (b) and (c) represent significant differences between CP and CP + RA100 treated group of animals. The treatment of CP caused a significant increase in the expression of p10 caspase-1 (***p < 0.001) and p20 caspase-1 (***p < 0.001) proteins when compared with control group of animals. However, the treatment of RA along with CP caused a significant restoration in the expression of p10 caspase-1 (^###p < 0.001) and p20 caspase-1 (^##p < 0.01) proteins when compared with CP alone treated group of mice. No significant differences were noticed in RA alone treated group of animals when compared with control group of animals. Data are expressed as means ± SEM (n = 6).

Figure 6

Western blotting analysis of inflammatory markers. Effect of RA and CP on the expression of COX-2 and NFκB-p65 in mouse kidney tissue as studied by western blot analysis is shown in (a) by immunoblots of inflammatory markers COX-2 and NFκB-p65. The administration of CP caused an increase in the expression of COX-2 and NFκB-p65 proteins when compared with control group of animals. While the treatment of RA along with the administration of CP decreases the expression of COX-2 and NFκB-p65 proteins when compared with CP alone treated group of mice. In this figure histograms (b) and (c) represent significant differences between CP and CP + RA100 treated group of animals. The treatment of CP caused a significant increase in the expression of COX2 (***p < 0.001) and NFκB-p65 (***p < 0.001) proteins when compared with control group of animals. However, the treatment of RA along with CP caused a significant restoration in the expression of COX-2 (^##p < 0.01) and NFκB-p65 (^#p < 0.05) proteins when compared with CP alone treated group of mice. No significant differences were noticed in RA alone treated group of animals when compared with control group of animals. Data are expressed as means ± SEM (n = 6).

Figure 7

Effect of RA and CP treatments on the expression levels of HMGB-1, TLR-4, MyD88 and IL-1R1 proteins. In figure histograms (a)–(d) represent significant changes in the expression level of HMBG-1, TLR-4, MyD88 and IL-1R1 proteins, respectively measured by ELISA. The administration of CP caused a significant increase in the level of HMBG-1 (***p < 0.001) (a), TLR-4 (***p < 0.001) (b), MyD88 (***p < 0.001) (c) and IL-1R1 (***p < 0.001) (d) at the dose of 20 mg/kg b.wt. in mouse kidney when compared with control group of animals. However, administration of RA at the dose of 100 mg/kg b.wt. caused a significant restoration of the expression level of HMBG-1 (^##p < 0.01), TLR-4 (^#p < 0.05), MyD88 (^##p < 0.01) and IL-1R1 (^#p < 0.05) protein levels when compared with CP alone group of animals. There was no significant change in RA alone treated animals when compared with control group of animals. Each value is represented as mean ± SEM (n = 6).

Figure 8

Summarized schematic pathway representation of NLRP3 inflammasome modulation by RA administration in CP-induced mouse AKI model. RA showed anti-inflammatory effects by inhibiting the proinflammatory markers in the mouse kidney.