Plasma Kallikrein as a Modulator of Liver Injury/Remodeling

Abstract

The occurrence and persistence of hepatic injury which arises from cell death and inflammation result in liver disease. The processes that lead to liver injury progression and resolution are still not fully delineated. The plasma kallikrein-kinin system (PKKS) has been shown to play diverse functions in coagulation, tissue injury, and inflammation, but its role in liver injury has not been defined yet. In this study, we have characterized the role of the PKKS at various stages of liver injury in mice, as well as the direct effects of plasma kallikrein on human hepatocellular carcinoma cell line (HepG2). Histological, immunohistochemical, and gene expression analyses were utilized to assess cell injury on inflammatory and fibrotic factors. Acute liver injury triggered by carbon tetrachloride (CCl₄) injection resulted in significant upregulation of the plasma kallikrein gene (Klkb1) and was highly associated with the high mobility group box 1 gene, the marker of cell death (r = 0.75, p < 0.0005, n = 7). In addition, increased protein expression of plasma kallikrein was observed as clusters around necrotic areas. Plasma kallikrein treatment significantly increased the proliferation of CCl₄-induced HepG2 cells and induced a significant increase in the gene expression of the thrombin receptor (protease activated receptor-1), interleukin 1 beta, and lectin-galactose binding soluble 3 (galectin-3) (p < 0.05, n = 4). Temporal variations in the stages of liver fibrosis were associated with an increase in the mRNA levels of bradykinin receptors: beta 1 and 2 genes (p < 0.05; n = 3-10). In conclusion, these findings indicate that plasma kallikrein may play diverse roles in liver injury, inflammation, and fibrosis, and suggest that plasma kallikrein may be a target for intervention in the states of liver injury.

Keywords: fibrosis; inflammation; kallikrein-kinin system; liver injury; necrosis; plasma kallikrein; remodeling.

FIGURE 1

Pronounced modification in the hepatic PKKS genes in acutely injured mice. (A) Schematic representation of carbon tetrachloride (CCl₄)-induced acute liver injury at days 1, 2, and 3 after the CCl₄ and mineral oil (vehicle) injection. (B) Representative H& E (original magnification ×40) staining of liver sections; necrotic areas are delineated by marked areas quantified using ImageJ software; data are represented as mean ± SEM (one-way ANOVA followed by Sidak’s multiple comparisons, *p < 0.05, ****p <0.0001; n = 7 mice per group). (C) Gene expression analysis of the PKKS (Klkb1, Kng1, F2r, and F2) and Hmgb1 genes. Data are shown as minimum to maximum values of box plots with whiskers extending 1.5 times the interquartile range. Center lines indicate the medians, while box limits represent the 25th and 75th percentiles (n = 7 per group). Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons, where **p <0.005, ***p <0.0005 (CCl₄ vs. Vehicle). (D) Association between Hmgb1 and Klkb1, F2r or Kng1 genes, correlative plots were assessed by the Spearman correlation.

FIGURE 2

Klkb1 gene expression is translated to the pre-kallikrein protein and stimulates cell proliferation. (A) Representative images of immunostaining of plasma kallikrein (black arrows; dark red clusters; original magnification ×40 (top) and ×100 (bottom)) at days 1, 2, and 3 of liver injury after the CCl₄ or vehicle (mineral oil) injection. (B) Percentage viability of HepG2 treated 1 or 2 mM CCl₄ or 2.5 ng/ml plasma kallikrein (PKK) for 24 h (n = 8). (C) Percentage viability and (D) proliferation of plasma kallikrein–treated cells for 1 or 2 days (n = 8). Data are shown as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons, where *p < 0.05, **p < 0.005 (PKK vs. control,), ***p < 0.0005 (1 mM CCl₄ vs. control; 2 mM CCl₄ vs. 1mM CCl₄), ****p < 0.0001 (Day 2-PKK vs. others; 1mM CCl₄+PKK vs. 1mM CCl₄). (E) Plasma kallikrein (PKK) increased the mRNA levels of F2R, and IL1B genes (n = 8). Data are shown as mean ± SEM. Statistical significance was determined by the Mann–Whitney test, *p < 0.05 (PKK versus CTL). CTL corresponds to the control group with 0.5 % DMSO as vehicle.

FIGURE 3

Strong association of PKKS genes to hepatic mRNA levels of inflammatory markers in acute liver injury. (A) Tnfa, Il1b, and Il6 gene induction implicates an inflammatory response to liver injury over the time course observed. (B) Spearman correlation studies between the Klklb1 gene and Ilb gene, the F2r gene and Il1b gene, and the Kng1 gene and Il1b gene. Data representation and statistical analysis were performed as described in the legend for Figure 1C (n = 7 per group). *p < 0.05, **p < 0.005 (CCl₄ vs. Vehicle), and correlative plots were assessed by the Spearman correlation. Vehicle corresponds to mineral oil.

FIGURE 4

Strong association of immune cells recruiting markers in acute liver injury. (A) Chemokine genes, Ccl2 and Ccl3; recruitment genes Adgre1, Ly6c, and Ly6g; and Mpo gene, a marker of neutrophil activation. (B) The correlation analysis by Spearman among the Klkb1 and Ccl3 genes; the Klkb1 and Adgre1 genes; the Klkb1 and Ly6c genes; and the Klkb1 and Mpo genes. Data representation and statistical analysis were performed as described in the legend for Figure 1C. Outliers are shown by dots outside the whiskers (n = 7 per group). *p < 0.05, **p < 0.005, ***p < 0.0005 (CCl₄ vs. Vehicle), and correlative plots were assessed by Spearman correlation. Vehicle corresponds to mineral oil.

FIGURE 5

Early induction of myofibroblast markers of activation in acute liver injury. (A) mRNA expression of Lgals3 and Ccn2. Data representation and statistical analysis were performed as described in the legend for Figure 1C. (n = 7 per group). Outliers are shown by dots outside the whiskers, *p < 0.05**p < 0.005, ***p < 0.0005 (CCl₄ vs. Vehicle). (B) Association of Klkb1, and Lgals3 and Ccn2, correlative plots were assessed by Spearman correlation. Vehicle corresponds to mineral oil. (C) Gene expression of LGALS3 and CCN2 in HepG2 cells were induced with plasma kallikrein (PKK). Data are shown as mean ± SEM (n = 4 per group), and statistical significance was determined by the Mann–Whitney test; *p < 0.05 (PKK vs. CTL). CTL corresponds to culture media only.

FIGURE 6

Upregulation of markers of fibrosis: inflammatory processes are regulated. (A) Schematic representation of CCl₄-induced chronic liver injury. Hepatic mRNA expression of (B) Lgals3, Ccn2, Timp1, Mmp9, and Mmp2; (C) inflammatory and chemokine genes, Tnfa, Il1b, Ccl2, and Ccl3; (D) the PKKS genes and thrombin (F2). Data representation for box plots was performed as described in the legend for Figure 1C (n = 5–6 mice per group); statistical significance was determined by the Mann–Whitney test, where *p < 0.05, **p < 0.005 (CCl₄ vs. Vehicle). Vehicle corresponds to mineral oil.

FIGURE 7

Klkb1 and Kng1 gene expressions at different time points of the chronic liver injury model. (A) Schematic representation of CCl₄-induced liver fibrosis at the 4th week. Similar pattern of mice injections was performed at the 6th and 7th weeks as well. (B) Varying representative images of collagen fibers stained with picro sirius red (stained red). (C) Quantification of histological staining by ImageJ software (data representation by mean ± SEM, n = 3–10 mice per group: one-way ANOVA followed by Sidak’s multiple comparisons; ****p < 0.0001). The quantified areas in each sample were normalized to their average time points of controls before statistical analysis. Hepatic mRNA expression (D, E, F) of Lgals3, Ccn2, and Klkb1; (G) plot analysis of Klkb1 gene and (H) Kng1 gene; (I) plot analysis of Kng1 gene and (J) F2 gene; (K) plot analysis of F2 gene. Data representation for box plots was performed as described in the legend for Figure 1C. Outliers are shown by dots outside the whiskers (n = 3–6 mice for Vehicle and 7–10 for CCl₄ treatment groups); statistical significance was determined by the Mann–Whitney test, where *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001 (CCl₄ vs. Vehicle). Vehicle corresponds to mineral oil.

FIGURE 8

PKKS receptors at different time points of the chronic injury model. (A, B) F2r and F2rl1 genes. (C, D) Plot analysis of the association among F2r, F2rl1, and Lgals3, Ccn2 genes, and fibrotic area (FA). Hepatic mRNA expression of (E, F) Bdkrb2 and Bdkrb1. (G, H) Plot analysis of the association among Bdkrb2, Bdkrb1, and Lgals3, Ccn2 genes, and fibrotic area (FA). Data representation for box plots was performed as described in the legend for Figure 1C. Outliers are shown by dots outside the whiskers (n = 3–6 mice for Vehicle and 7–10 for CCl₄ treatment groups); statistical significance was determined by the Mann–Whitney test, where *p < 0.05; **p < 0.005; ***p < 0.0005 (CCl₄ vs. Vehicle) Vehicle corresponds to mineral oil.

FIGURE 9

Plasma kallikrein-kinin system diverse roles in liver injury.