Reversing established sepsis in rats with human vasoactive hormone adrenomedullin and its binding protein

Abstract

We recently demonstrated that early administration of rat adrenomedullin (AM), a vasoactive peptide, in combination with its binding protein (human AMBP-1) produces various beneficial effects in sepsis. Human AM is a 52-amino acid peptide, but rat AM differs from human AM, having only 50 amino acid residues, with two amino acid deletions and six substitutions. It remains unknown whether a combination of human AM and human AMBP-1 (AM/AMBP-1) is also beneficial in sepsis and, if so, whether human AM/AMBP-1 reverses established sepsis in rats. To test the effects of human AM/AMBP-1, we induced sepsis in male adult rats by cecal ligation and puncture (CLP). At 10 h after CLP (i.e., severe sepsis), human AM (12-48 microg/kg body weight) was administered in combination with human AMBP-1 (40-160 microg/kg body weight). Vehicle-treated animals received a nonspecific human plasma protein (albumin). Blood and intestinal samples were collected at 20 h for various measurements. In additional groups of septic animals, the gangrenous cecum was surgically excised at 20 h after CLP. The 10-day survival was recorded. Our results showed that tissue injury, as evidenced by increased levels of transaminases and lactate, was present at 20 h after CLP. Proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 were significantly elevated. Gut barrier dysfunction, manifested by increased mucosal permeability to hydrophilic macromolecules and increased bacterial translocation to mesenteric lymph nodes, also occurred at 20 h after CLP. Administration of human AM/AMBP-1 in established sepsis markedly attenuated tissue injury, reduced proinflammatory cytokine levels, ameliorated intestinal-barrier dysfunction, and improved the survival rate from 47% to 67%-80%. Thus, human AM/AMBP-1 can be further developed as a safe and effective therapy for patients with established sepsis.

Figure 1

Alterations in circulating levels of (A) aspartate aminotransferase (AST) and (B) alanine aminotransferase (ALT) in sham-operated animals (Sham) and septic animals treated with human albumin (Vehicle) or human adrenomedullin (AM)/human adrenomedullin binding protein (AMBP)-1 (12/40, 24/80, or 48/160 μg/kg BW) at 20 h after cecal ligation and puncture (CLP). Data are presented as means ± SE (n = 4–6/group) and compared by one-way analysis of variance and Student-Newman-Keuls test: *P < 0.05 versus sham group; ^#P < 0.05 versus vehicle group.

Figure 2

Alterations in circulating levels of lactate in sham-operated animals (Sham) and septic animals treated with human albumin (Vehicle) or human adrenomedullin (AM)/human adrenomedullin binding protein (AMBP)-1 (12/40, 24/80, or 48/160 μg/kg BW) at 20 h after cecal ligation and puncture (CLP). Data are presented as means ± SE (n = 5–6/group) and compared by one-way analysis of variance and Student-Newman-Keuls test: *P < 0.05 versus sham group; ^#P < 0.05 versus vehicle group.

Figure 3

Alterations in circulating levels of (A) TNF-α and (B) IL-6 in sham-operated animals (Sham) and septic animals treated with human albumin (Vehicle) or human adrenomedullin (AM)/human adrenomedullin binding protein (AMBP)-1 (12/40, 24/80, or 48/160 μg/kg BW) at 20 h after cecal ligation and puncture (CLP). Data are presented as means ± SE (n = 6/group) and compared by one-way analysis of variance and Student-Newman-Keuls test: *P < 0.05 versus sham group; ^#P < 0.05 versus vehicle group.

Figure 4

Alterations in intestinal mucosal permeability (A) to fluorescein isothiocyanate dextran with a molecular weight of 4000 Da (FD4) and bacterial translocation to mesenteric lymph nodes (B) in sham-operated animals (Sham) and septic animals treated with human albumin (Vehicle) or human adrenomedullin (AM)/human adrenomedullin binding protein (AMBP)-1 (24/80 μg/kg BW) at 20 h after cecal ligation and puncture (CLP). Data are presented as means ± SE (n = 4–8/group) and compared by one-way analysis of variance and Student-Newman-Keuls test: *P < 0.05 versus sham group; ^#P < 0.05 versus vehicle group.

Figure 5

Alterations in the survival rate at 10 d after cecal ligation and puncture and cecal excision with human albumin treatment (CLPE+Vehicle) or human adrenomedullin (AM)/human adrenomedullin binding protein (AMBP)-1 (12/40, 24/80, or 48/160 μg/kg BW) treatment. There were 17–21 animals in each group. The survival rates were estimated by the Kaplan–Meier method and compared by using the log-rank test. *P < 0.05 versus vehicle group.