Rhein prevents endotoxin-induced acute kidney injury by inhibiting NF-κB activities

Abstract

This study aimed to explore the effect and mechanisms of rhein on sepsis-induced acute kidney injury by injecting lipopolysaccharide (LPS) and cecal ligation and puncture (CLP) in vivo, and on LPS-induced HK-2 cells in vitro. For histopathological analysis, rhein effectively attenuated the severity of renal injury. Rhein could significantly decrease concentration of BUN and SCr and level of TNF-α and IL-1β in two different mouse models of experimental sepsis. Moreover, rhein could markedly attenuate circulating leukocyte infiltration and enhance phagocytic activity of macrophages partly impaired at 12 h after CLP. Rhein could enhance cell viability and suppresse the release of MCP-1 and IL-8 in LPS-stimulated HK-2 cells Furthermore, rhein down regulated the expression of phosphorylated NF-κB p65, IκBα and IKKβ stimulated by LPS both in vivo and in vitro. All these results suggest that rhein has protective effects on endotoxin-induced kidney injury. The underlying mechanism of rhein on anti-endotoxin kidney injury may be closely related with its anti-inflammatory and immunomodulatory properties by decreasing NF-κB activation through restraining the expression and phosphorylation of the relevant proteins in NF-κB signal pathway, hindering transcription of NF-κB p65.These evidence suggest that rhein has a potential application to treat endotoxemia-associated acute kidney injury.

Figure 1. The chemical structure of rhein.

(C₁₅H₈O₆, molecular weight = 284.22).

Figure 2. Effect of rhein on kidney injury after LPS administration.

Representative histological changes in kidneys obtained from mice of different groups (A) Control group; (B) LPS group; (C) Rhein (20 mg/kg) + LPS group; (D) Rhein (40 mg/kg) + LPS group; (E) Rhein (80 mg/kg) + LPS group.The sections shown were harvested 12 h after LPS injection and stained with H&E. Magnification: ×400. (F)Pathological score of representative kidney samples of each group. Data are represented as mean ± SD of 5 animals of each group. ###P < 0.001 versus control group; **P < 0.01 and ***P < 0.001 versus LPS group.

Figure 3

Effects of rhein on serum BUN (A) and SCr(B). Data are represented as mean ± SD of 10 animals of each group. ###p < 0.001 compared to control group; **p < 0.01 and ***p < 0.001 compared to LPS group.

Figure 4

The effect of rhein on TNF-α (A) and IL-1β (B) levels after LPS challenge. Quantitation of TNF-α and IL-1β in renal tissue was performed by ELISA. Data are represented as mean ± SD of 10 animals of each group. ###p < 0.001 compared to control group; *p < 0.05, **p < 0.01 and ***p < 0.001 compared to LPS group.

Figure 5. Effect of rhein on phospho-NF-κB p65 localization and expression in AKI by immunohistochemistry (magnification × 400).

(A) Control group; (B) LPS group; (C) Rhein (20 mg/kg) + LPS group; (D)Rhein (40 mg/kg) + LPS group. (E) Rhein (80 mg/kg) + LPS group. (F) IOD values of phospho-NF-κB p65 staining. Data are represented as mean ± SD of 5 animals of each group, ### P < 0.001 versus control group; **p < 0.01 and ***P < 0.001 versus LPS group.

Figure 6. Effects of rhein on the expression of NF-κB p65, p-IκBα and phospho-IKKβ in LPS-induced AKI.

Protein extracts were obtained from kidney tissues and proteins expression level was detected by Western blotting analysis. All data represent the means ± SD from three separate experiments. ##p < 0.01 and ###p < 0.001 compared to control group; **p < 0.01 compared to LPS group.

Figure 7

Effects of rhein on serum BUN (A) and SCr(B) in CLP model. Data are represented as mean ± SD of 10 animals of each group. ###p < 0.001 compared to sham group; **p < 0.01, ***p < 0.001 compared to CLP group.

Figure 8

Effects of rhein on TNF-α (A) and IL-1β (B) levels after CLP challenge. Quantitation of TNF-α and IL-1β in renal tissue was performed by ELISA. Data are represented as mean ± SD of 10 animals of each group. ###p < 0.001 compared to sham group; *p < 0.05, **p < 0.01 and ***p < 0.001 compared to CLP group.

Figure 9. The effect of rhein on peripheral white blood cell counts in CLP-induced sepsis. Blood samples were withdrawn by cardiac puncture with a heparinized syringe at 12 h after the CLP surgery.

total and differential cell counts were measured. Data are represented as mean ± SD of 10 animals of each group. #p < 0.05 and ##p < 0.01 compared to sham group; *p < 0.05, **p < 0.01 and ***p < 0.001 compared to CLP group.

Figure 10. The effect of rhein on peritoneal macrophage phagocytic activity in CLP-induced mice sepsis.

Macrophages harvested 12 h after CLP were incubated with zymosan and NBT. Phagocytosis was measured as OD 630 nm. Data are expressed as mean ± SD (n = 10). ##p < 0.01 compared to sham group; *p < 0.05,**p < 0.01 and ***p < 0.001 compared to CLP group.

Figure 11. The effect of rhein on HK-2 cells viability were tested by MTT assay.

(A)Effect of rhein on HK-2 cells proliferation in normal condition by MTT assay.(B) Effect of rhein on LPS-induced HK-2 cells proliferation by MTT assay. Results are expressed as percentage of viable cells when compared with control groups. Data are expressed as mean ± SD. ##p < 0.01 vs. control group, *p < 0.05 and **p < 0.01 vs. LPS alone.

Figure 12

The effect of rhein on MCP-1 (A) and IL-8 (B) release induced by LPS in HK-2 cells. Cells were treated with LPS with or without rhein (10, 20, and 40 μM) for 24 h. 100 μl of culture medium in each group was taken out to measure the levels of MCP-1 and IL-8 using ELISA kits. Data are represented as mean ± SD of three independent experiments. ###p < 0.001 vs. control group, *p < 0.05, **p < 0.01 vs. LPS alone.

Figure 13. The effect of rhein on NF-κB signal pathway in LPS-induced HK-2 cells.

The expression of NF-κB p65, p-IκBα and phospho-IKKβ were assessed by Western blot analysis. β-actin was used as an internal control. Results are expressed as fold increase over control group. Data are represented as mean ± SD of three independent experiments. ##p < 0.01 vs. control group, **p < 0.01 vs. LPS alone.