Plasma Kallikrein-Kinin system mediates immune-mediated renal injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a major environmental pollutant. An immunological response is a newly-recognized mechanism for TCE-induced kidney damage. However, the role of the plasma kallikrein-kinin system (KKS) in immune-mediated kidney injury has never been examined. This study aimed to explore the role of the key components of the KKS, i.e. plasma kallikrein (PK), bradykinin (BK) and its receptors B1R and B2R, in TCE-induced kidney injury. A mouse model of skin sensitization was used to explore the mechanism of injury with or without a PK inhibitor PKSI. Kidney function was evaluated by measuring blood urea nitrogen (BUN) and creatinine (Cr) in conjunction with histopathologic characterization. Plasma BK was determined by ELISA; Renal C5b-9 membrane attack complex was evaluated by immunohistochemistry. Expression of BK and PK in the kidney was detected by immunofluorescence. mRNA and protein levels of B1R and B2R were assessed by real-time qPCR and Western blot. As expected, numerous inflammatory cell infiltration and tubular epithelial cell vacuolar degeneration were observed in TCE-sensitized mice. Moreover, serum BUN and Cr and plasma BK were increased. In addition, deposition of BK, PK and C5b-9 were observed and B1R and B2R mRNA and proteins levels were up-regulated. Pre-treatment with PKSI, a highly selective inhibitor of PK, alleviated TCE-induced renal damage. In addition, PKSI attenuated TCE-induced up-regulation of BK, PK and its receptors and C5b-9. These results provided the first evidence that activation of the KKS contributed to immune-mediated renal injury induced by TCE and also helped to identify the KKS as a potential therapeutic target for mitigating chemical sensitization-induced renal damage.

Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals.

[Study on the expression of bradykinin and its receptors B1R and B2R in the kidney immune injury in trichloroethylene-sensitized mouse].

OBJECTIVE: To study the expression of bradykinin and its receptors B1R and B2R in the kidney immune injury in trichloroethylene-sensitized mouse and discuss the pathogenesis of Dermatitis Medicamentosa-like of TCE (ODMLT). METHODS: On the first days, intradermal injection by 50% TCE and the amount of FCA mixture 100 µl for initial sensitization; on 4, 7, 10 days, painted abdominal skin by 100 µl 50% TCE for three sensitization, on 17, 19 days, painted on the back skin by 100 µl 30% TCE for initial excitation and the last challenge; 24 h before each challenge, PKSI-527+TCE group received intraperitoneal injection by inhibitor PKSI-527 (50 mg/kg); solvent control group treat without TCE and sensitization and excitation reagent the same proportion of olive oil and acetone mixture, blank control group without any treatment. Before killing the mouse, renal weight and body weight were recorded. The renals and plasma were separated at 24 h, 48 h, 72 h and 7 d after the last challenge and observed pathological of the renals. Expression of B1R and B2R in renal were examined by immunofluorescence technique. Plasma were examined by ELISA for BK. RESULTS: The renal pathological examination revealed the apparent damage of TCE sensitized mice which compared to solvent control group showed obvious cellular infiltration, vacuolar degeneration of renal tubular epithelial cells. The renal damage of PKSI-527+TCE-sensitized groups which compared to the corresponding point of TCE-sensitized groups showed significantly reduced. The expression of BK in 24 h, 48 h and 72 h TCE-sensitized groups were significant higher than solvent control group and related TCE non-sensitized groups (P < 0.05) and 72 h point compared to the corresponding point of PKSI-527+TCE group was also increased, the difference was statistically significant (P < 0.05). The expression levels of B1R and B2R in the kidney in 24 h, 48 h, 72 h and 7 d TCE-sensitized groups were obviously higher than solvent control group and related TCE non-sensitized groups. The expression levels of B1R and B2R in the kidney in the four point of PKSI-527+TCE sensitized group were relatively lower than the corresponding point of TCE sensitized group. CONCLUSION: KKS activation may involved in the renal immune injury of trichloroethylene-sensitized mouse and the expression change of bradykinin and its receptors B1R and B2R which may play an important role in the process.

An Animal Model of Trichloroethylene-Induced Skin Sensitization in BALB/c Mice.

Trichloroethylene (TCE) is a major occupational hazard and environmental contaminant that can cause multisystem disorders in the form of occupational medicamentosa-like dermatitis. Development of dermatitis involves several proinflammatory cytokines, but their role in TCE-mediated dermatitis has not been examined in a well-defined experimental model. In addition, few animal models of TCE sensitization are available, and the current guinea pig model has apparent limitations. This study aimed to establish a model of TCE-induced skin sensitization in BALB/c mice and to examine the role of several key inflammatory cytokines on TCE sensitization. The sensitization rate of dorsal painted group was 38.3%. Skin edema and erythema occurred in TCE-sensitized groups, as seen in 2,4-dinitrochlorobenzene (DNCB) positive control. Trichloroethylene sensitization-positive (dermatitis [+]) group exhibited increased thickness of epidermis, inflammatory cell infiltration, swelling, and necrosis in dermis and around hair follicle, but ear painted group did not show these histological changes. The concentrations of serum proinflammatory cytokines including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2 were significantly increased in 24, 48, and 72 hours dermatitis [+] groups treated with TCE and peaked at 72 hours. Deposition of TNF-α, IFN-γ, and IL-2 into the skin tissue was also revealed by immunohistochemistry. We have established a new animal model of skin sensitization induced by repeated TCE stimulations, and we provide the first evidence that key proinflammatory cytokines including TNF-α, IFN-γ, and IL-2 play an important role in the process of TCE sensitization.

[Expression of C3aR and C5aR in trichloroethylene-sensitized mouse liver].

OBJECTIVE: To study the expression of C3aR and C5aR in trichloroethylene-sensitized mouse liver injury and discuss the pathogenesis of Dermatitis Medicamentosa-like of TCE (DMLT). METHODS: 6∼8 w female BALB/c mouse were randomly divided into blank control group, solvent control group and TCE treatment group. TCE was given to the mouse for sensitization at 1th, 4th, 7th, 10th day and challenge at 17th day and 19th day. Before killing mouse, liver weight and body weight were recorded. The livers were separated at 24 h, 48 h, 72 h and 7 d after challenge. And the liver sections were used for immunofluorescence stain and RT-PCR to detect the expression levels of C3aR and C5aR. RESULTS: Microscopic examination showed no significant change in liver structure or organization in TCE non-sensitized group, while liver cell oedema, cell necrosis and inflammatory cell infiltration were clearly observed in TCE-sensitized groups. The expression levels of C3aR and C5aR in 24 h, 48 h, 72 h and 7 d TCE-sensitized groups were significant higher than blank control group, solvent control group and related TCE non-sensitized groups (P < 0.05). CONCLUSION: Complement activation was involved in TCE-induced liver injury and C3aR and C5aR might play essential role in the process.

[Levels of complement components C3a and C5a in renal injury among trichloroethylene-sensitized BALB/c mice].

OBJECTIVE: To determine the levels of complement components C3a and C5a in the kidneys of trichloroethylene (TCE)-sensitized BALB/c mice, and to investigate the role of complement components in TCE-induced renal injury among BALB/c mice. METHODS: Sixty-two female BALB/c mice were randomly divided into blank control group, vehicle control group, and TCE sensitization group. The mice in TCE sensitization group were sensitized by one intracutaneous injection and one abdominal smear of TCE. At 24 h, 48 h, 72 h, and 7 d after the second sensitization, mice were sacrificed, and the blood and kidneys were collected. An automatic biochemical analyzer was used in the determination of serum blood urea nitrogen (BUN) and creatinine (Cr). The levels of C3a and C5a in the kidneys were determined by immunohistochemistry. RESULTS: The sensitization rate of TCE sensitization group was 42.0%. Kidney coefficient and serum levels of BUN and Cr were significantly increased in the TCE sensitization group as compared with the vehicle control group at 48 h and 72 h after sensitization (P < 0.05). The kidney coefficients of the TCE sensitization group at 48 h and 72 h were significantly higher than those of the control groups (P < 0.05). In comparison with the vehicle control group, however, no significant change was found in kidney coefficient, serum BUN, or serum Cr at 7 d after TCE sensitization (P > 0.05). Levels of C3a and C5a at 48 h (3.80±0.84 and 4.00±1.00, respectively) and 72 h (4.40 ± 1.14 and 4.40 ± 1.14, respectively) after sensitization were all significantly higher than those of the vehicle control group (P < 0.05), but no significant difference was found in level of C3a (1.80±0.45) or C5a (2.00 ± 0.71) at 7 d (P > 0.05). CONCLUSION: TCE sensitization can induce renal injury in mice. Levels of complement components C3a and C5a are elevated in the kidneys of sensitized mice, indicating that C3a and C5a may be involved in the renal injury induced by TCE sensitization.

Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.

Complement activation and liver impairment in trichloroethylene-sensitized BALB/c mice.

Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.