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. 2018 Dec;15(1):63-72.
doi: 10.1080/1547691X.2017.1420712.

NF-κB signaling pathway-enhanced complement activation mediates renal injury in trichloroethylene-sensitized mice

Min Liu  1 , Hui Wang  2   3 , Jiaxiang Zhang  1   3 , Xiaodong Yang  1 , Bodong Li  1 , Changhao Wu  4 , Qixing Zhu  3
Affiliations

NF-κB signaling pathway-enhanced complement activation mediates renal injury in trichloroethylene-sensitized mice

Min Liu et al. J Immunotoxicol. 2018 Dec.

Abstract

Both NF-κB pathway and complement activation appear to be involved in kidney damage induced by trichloroethylene (TCE). However, any relationship between these two systems has not yet been established. The present study aimed to clarify the role of NF-κB in complement activation and renal injury in TCE-sensitized BALB/c mice. Mice were sensitized by an initial subcutaneous injection and repeated focal applications of TCE to dorsal skin at specified timepoints. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was injected (intraperitoneal) before the final two focal TCE challenges. In the experiments, mice had their blood and kidneys collected. Kidney function was evaluated via blood urea nitrogen (BUN) and creatinine (Cr) content; renal histology was examined using transmission electron microscopy (TEM). Kidney levels of phospho-p65 were assessed by Western blot and kidney mRNA levels of interleukin (IL)-1β, IL-6, IL-17, tumor necrosis factor (TNF)-α, and p65 by real-time quantitative PCR. Presence of C3 and C5b-9 membrane attack complexes in the kidneys was evaluated via immunohistochemistry. The results showed there was significant swelling, vacuolar degeneration in mitochondria, shrinkage of microvilli, disappearance of brush borders, segmental foot process fusion, and glomerular basement membrane thickening (or disrobing) in kidneys from TCE-sensitized mice. In conjunction with these changes, serum BUN and Cr levels were increased and IL-1β, IL-6, IL-17, and TNFα mRNA levels were elevated. Levels of p65 and phospho-p65 protein were also up-regulated, and there was significant C3 and C5b-9 deposition. PDTC pretreatment attenuated TCE-induced up-regulation of p65 and its phosphorylation, complement deposition, cytokine release, and renal damage. These results provide the first evidence that NF-κB pathway has an important role in TCE-induced renal damage mediated by enhanced complement activation in situ.

Keywords: NF-κB; Trichloroethylene; p65; renal injury.

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Figures

Figure 1.
Figure 1.
Pathways involved in NF-κB and the complement system. IκKα = IκB kinase that phosphorylates IκKα; IκB = κB protein inhibitor; p50 and p65 = NF-κB family members; p-p65 = phosphorylated p65; C3 = Complement 3.
Figure 2.
Figure 2.
Protocol for TCE induction of sensitization in mice. After 7 days of adaptive feeding, TCE was applied to the abdomen by intradermal injection. On Days 4, 7, and 10, TCE (100 μl of 30% TCE [TCE:olive oil:acetone, 3:2:5 {v/v/v}] solution) was applied to the dorsal skin. Animals were then re-challenged with TCE on Days 17 and 19. NF-κB inhibitor PDTC was applied to the PDTC pre-treatment group of mice 24 hr before each challenge.
Figure 3.
Figure 3.
Ultrastructure changes of renal tissue as monitored by transmission electron microscopy. (A) Control. (B) Solvent control. (C) TCE+, with basement membrane thickening, foot process fusions, swelling and vacuolar degeneration of mitochondria. (D) TCE-, with basement membrane thickening. (E) PDTC+, basement membrane thickening, foot process fusions. (F) PDTC-, with basement membrane thickening, foot process fusions. Black arrows indicate podo-cytes and mitochondrial vacuolar degeneration, and glomerular basement membrane thickening. Changes noted in (C) were alleviated in (E). Magnification = 20,000X.
Figure 4.
Figure 4.
Serum BUN and Cr levels in the study mice. (A) BUN. (B) Cr. Value significantly different vs. asolvent control, bcorresponding sensitization-negative group, or c corresponding sensitization-positive group (each p < 0.05).
Figure 4.
Figure 4.
Serum BUN and Cr levels in the study mice. (A) BUN. (B) Cr. Value significantly different vs. asolvent control, bcorresponding sensitization-negative group, or c corresponding sensitization-positive group (each p < 0.05).
Figure 5.
Figure 5.
C3 deposition in mouse renal tissue. (A) Control. (B) Solvent control. (C) TCE+. (D) TCE−. (E) PDTC+. (F) PDTC−. Black arrows indicate deposition of C3. Magnification = 200×.
Figure 6.
Figure 6.
C5b-9 deposition in mouse renal tissue. (A) Control; (B) solvent control; (C) TCE+; (D) TCE−; (E) PDTC+；(F) PDTC−. Black arrows indicate C5b-9 deposition. Magnification = 200X.
Figure 7.
Figure 7.
Renal p65 gene and p-p65 protein expression. (A) p65 mRNA. Values significantly different vs. asolvent control or bTCE+ (each p < 0.05). (B) p-p65 expression. (C) Result expressed as ratio of p-p65:GAPDH. Values shown are means ± SD. Significant vs. asolvent control, or bTCE+ 72 hr group (each p < 0.05).
Figure 7.
Figure 7.
Renal p65 gene and p-p65 protein expression. (A) p65 mRNA. Values significantly different vs. asolvent control or bTCE+ (each p < 0.05). (B) p-p65 expression. (C) Result expressed as ratio of p-p65:GAPDH. Values shown are means ± SD. Significant vs. asolvent control, or bTCE+ 72 hr group (each p < 0.05).
Figure 7.
Figure 7.
Renal p65 gene and p-p65 protein expression. (A) p65 mRNA. Values significantly different vs. asolvent control or bTCE+ (each p < 0.05). (B) p-p65 expression. (C) Result expressed as ratio of p-p65:GAPDH. Values shown are means ± SD. Significant vs. asolvent control, or bTCE+ 72 hr group (each p < 0.05).
Figure 8.
Figure 8.
IL-1β, IL-6, IL-17, and TNFα gene expression in kidney. Expression levels were normalized against GAPDH. (A) IL-1β. (B) IL-6 (C) IL-17 (D) TNFα. Data shown are means ± SD. Value significantly different vs. asolvent control, bcorresponding sensitization-negative group, or vs. c corresponding sensitization-positive group (each p < 0.05).
Figure 8.
Figure 8.
IL-1β, IL-6, IL-17, and TNFα gene expression in kidney. Expression levels were normalized against GAPDH. (A) IL-1β. (B) IL-6 (C) IL-17 (D) TNFα. Data shown are means ± SD. Value significantly different vs. asolvent control, bcorresponding sensitization-negative group, or vs. c corresponding sensitization-positive group (each p < 0.05).
Figure 8.
Figure 8.
IL-1β, IL-6, IL-17, and TNFα gene expression in kidney. Expression levels were normalized against GAPDH. (A) IL-1β. (B) IL-6 (C) IL-17 (D) TNFα. Data shown are means ± SD. Value significantly different vs. asolvent control, bcorresponding sensitization-negative group, or vs. c corresponding sensitization-positive group (each p < 0.05).
Figure 8.
Figure 8.
IL-1β, IL-6, IL-17, and TNFα gene expression in kidney. Expression levels were normalized against GAPDH. (A) IL-1β. (B) IL-6 (C) IL-17 (D) TNFα. Data shown are means ± SD. Value significantly different vs. asolvent control, bcorresponding sensitization-negative group, or vs. c corresponding sensitization-positive group (each p < 0.05).
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