Kallistatin treatment attenuates lethality and organ injury in mouse models of established sepsis

Abstract

Introduction: Kallistatin levels in the circulation are reduced in patients with sepsis and liver disease. Transgenic mice expressing kallistatin are resistant to lipopolysaccharide (LPS)-induced mortality. Here, we investigated the effect of kallistatin on survival and organ damage in mouse models of established sepsis.

Methods: Mice were rendered septic by cecal ligation and puncture (CLP), or endotoxemic by LPS injection. Recombinant human kallistatin was administered intravenously six hours after CLP, or intraperitoneally four hours after LPS challenge. The effect of kallistatin treatment on organ damage was examined one day after sepsis initiation, and mouse survival was monitored for four to six days.

Results: Human kallistatin was detected in mouse serum of kallistatin-treated mice. Kallistatin significantly reduced CLP-induced renal injury as well as blood urea nitrogen, serum creatinine, interleukin-6 (IL-6), and high mobility group box-1 (HMGB1) levels. In the lung, kallistatin decreased malondialdehyde levels and HMGB1 and toll-like receptor-4 (TLR4) synthesis, but increased suppressor of cytokine signaling-3 (SOCS3) expression. Moreover, kallistatin attenuated liver injury, serum alanine transaminase (ALT) levels and hepatic tumor necrosis factor-α (TNF-α) synthesis. Furthermore, delayed kallistatin administration improved survival in CLP mice by 38%, and LPS-treated mice by 42%. In LPS-induced endotoxemic mice, kallistatin attenuated kidney damage in association with reduced serum creatinine, IL-6 and HMGB1 levels, and increased renal SOCS3 expression. Kallistatin also decreased liver injury in conjunction with diminished serum ALT levels and hepatic TNF-α and TLR4 expression. In cultured macrophages, kallistatin through its active site increased SOCS3 expression, but this effect was blocked by inhibitors of tyrosine kinase, protein kinase C and extracellular signal-regulated kinase (ERK), indicating that kallistatin stimulates a tyrosine-kinase-protein kinase C-ERK signaling pathway.

Conclusions: This is the first study to demonstrate that delayed human kallistatin administration is effective in attenuating multi-organ injury, inflammation and mortality in mouse models of polymicrobial infection and endotoxemia. Thus, kallistatin therapy may provide a promising approach for the treatment of sepsis in humans.

Figure 1

Delayed kallistatin treatment reduces renal injury and decreases blood urea nitrogen (BUN), serum creatinine, interleukin-6 (IL-6) and high mobility group box-1 (HMGB1) levels in cecal ligation and puncture (CLP) mice. (A) H&E staining was performed on kidney sections to examine renal histology (n = 4). The representative sections are shown at ×200 magnification. Levels of (B) BUN, (C) serum creatinine, (D) IL-6 and (E) HMGB1 were significantly decreased by kallistatin administration in CLP mice (n = 3 to 4). Data are expressed as means ± SE. *P <0.05 versus sham group; ^# P <0.05 versus CLP control group.

Figure 2

Delayed kallistatin treatment reduces malondialdehyde (MDA), high mobility group box-1 (HMGB1) and toll-like receptor-4 (TLR4) expression, and increases suppressor of cytokine signaling-3 (SOCS3) expression in lung tissue of cecal ligation and puncture (CLP) mice. Levels of (A) MDA, (B) HMGB1 and (C) TLR4 were significantly reduced in kallistatin-treated mice (n = 3 to 4). (D) Kallistatin administration also significantly increased SOCS3 expression compared to the sham group (n = 3). Data are expressed as means ± SE. *P <0.05 versus sham group; ^# P <0.05 versus CLP control group.

Figure 3

Delayed kallistatin administration attenuated liver injury, serum alanine transaminase (ALT) levels and liver tumor necrosis factor-α (TNF-α) expression in cecal ligation and puncture (CLP) mice. (A) H&E staining was performed on liver sections to examine liver histology (n = 4). Representative sections are shown at ×200 magnification. (B) Serum ALT levels and (C) liver TNF-α expression were significantly decreased in kallistatin-treated CLP mice (n = 3 to 4). Data are expressed as means ± SE. *P <0.05 versus sham group; ^# P <0.05 versus CLP control group.

Figure 4

Delayed kallistatin treatment improves survival in cecal ligation and puncture (CLP) mice and lipopolysaccharide (LPS)-induced endotoxemic mice. (A) Mice receiving human kallistatin (20 mg/kg, KS20) presented significantly reduced mortality during the observation period compared to CLP control mice (PBS group); sham-operated mice exhibited 100% survival (n = 16). (B) Mice receiving human kallistatin (20 mg/kg, KS20) exhibited significantly reduced mortality rate during the observation period compared to LPS control mice (n = 16). *P <0.05 versus CLP control group or LPS control group.

Figure 5

Delayed kallistatin treatment reduces renal injury, serum creatinine, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and high mobility group box-1 (HMGB1) levels, and increases renal suppressor of cytokine signaling-3 (SOCS3) expression in endotoxemic mice. (A) H&E staining was performed on kidney sections to examine histology (n = 4). The representative sections are shown at ×200 magnification. (B) Serum creatinine levels were attenuated by kallistatin treatment. (C) TNF-α, (D) IL-6 and (E) HMGB1 levels in serum of kallistatin-treated mice were significantly lower than that of the lipopolysaccharide (LPS) control mice. (F) Kallistatin treatment also significantly increased renal SOCS3 expression (n = 3 to 6). Data are expressed as means ± SE. *P <0.05 versus control group; ^# P <0.05 versus LPS control group.

Figure 6

Kallistatin treatment ameliorates liver injury, attenuates serum alanine transaminase (ALT) levels, and reduces tumor necrosis factor-α (TNF-α) and toll-like receptor-4 (TLR4) expression in liver tissue of LPS-induced endotoxemic mice. (A) H&E staining was performed on liver sections to examine histology (n = 4). The representative sections are shown at ×200 magnification. (B) Kallistatin administration reduces serum ALT levels (n = 3 to 4). Liver mRNA levels of (C) TNF-α and (D) TLR4 were significantly decreased by kallistatin treatment (n = 3). *P <0.05 versus control group; ^# P <0.05 versus LPS control group.

Figure 7

Kallistatin treatment enhances suppressor of cytokine signaling-3 (SOCS3) expression through activation of a tyrosine kinase-protein kinase C (PKC)-extracellular signal-regulated kinase (ERK) signaling pathway in RAW264.7 cells. (A) Macrophages were incubated with 0.05 μM wild-type kallistatin (WT-KS), heparin-mutant kallistatin (HM-KS) or active site-mutant kallistatin (AM-KS) for 12 hours. Wild-type or heparin mutant kallistatin, but not active site mutant kallistatin, significantly increased SOCS3 expression (n = 3). (B) The effect of kallistatin on SOCS3 expression in macrophages was blocked by PD98059 (PD; ERK inhibitor), chelerythrine (CHE; PKC inhibitor) and genistein (GEN; tyrosine kinase inhibitor). (C) ERK phosphorylation induced by kallistatin in macrophages was also reversed by PD98059, chelerythrine and genistein (n = 3). *P <0.05 versus control group; ^# P <0.05 versus kallistatin alone.

Figure 8

Proposed mechanisms mediated by kallistatin in sepsis-induced inflammation, organ injury and mortality. Kallistatin, via its heparin-binding domain, antagonizes TNF-α- and HMGB1-mediated inflammatory gene expression, and its active site is essential for inducing SOCS3 expression. HMGB1, high mobility group box-1; SOCS3, suppressor of cytokine signaling-3; TNF-α, tumor necrosis factor-α.