Kallikrein inhibitors limit kinin B(2) antagonist-induced progression of oedematous to haemorrhagic pancreatitis in rats

Abstract

Background and purpose: Exocrine hyperstimulation with caerulein is an established model for oedematous acute pancreatitis. Prevention of oedema formation by bradykinin B(2) receptor antagonists induces a progression to a haemorrhagic course in this model. We have investigated whether increased kallikrein activity in the pancreas is responsible for vascular damage and whether this could be prevented by selective kallikrein inhibitors.

Experimental approach: Caerulein was infused i.v. and vascular damage was assessed by histological evaluation and determination of haemoglobin accumulation in the tissue. In addition, oedema formation, tissue and plasma kallikrein (PK) activities and the endogenous kallikrein inhibitors alpha(1)-antitrypsin (alpha(1)-AT) and alpha(2)-macroglobulin (alpha(2)-M) were measured.

Key results: Haemorrhagic lesions induced by icatibant in caerulein-induced pancreatitis were associated with a reduction in alpha(1)-AT and alpha(2)-M in the pancreas and a concomitant augmentation of tissue kallikrein (TK) activity. The TK inhibitor VA999024 (previously FE999024), or its combination with the PK inhibitor VA999026 (previously FE999026), inhibited oedema formation to the same extent but did not induce vascular damage. Furthermore, VA999024 inhibited TK activity. When icatibant was combined with VA999024 and VA999026, progression from oedematous to haemorrhagic pancreatitis was abolished.

Conclusions and implications: Reduced oedema formation by B(2) antagonists prevented influx of endogenous kallikrein inhibitors and led to an excessive activity of kallikrein in the pancreas leading to vascular damage. This can be prevented by a combined inhibition of both tissue-type and plasma-type kallikrein. Kallikrein inhibitors thus should be further evaluated for their therapeutic potential in preventing haemorrhagic lesions in acute pancreatitis.

Figure 1

Effects of the B₂ antagonist icatibant (ICAT), the TK inhibitor VA999024 (TKI) and the PK inhibitor VA999026 (PKI) in caerulein (CRL)-induced pancreatitis. (a) Oedema formation and (b) haemoglobin accumulation in the pancreas: CRL or phosphate-buffered saline (PBS) was infused i.v; icatibant (100 nmol kg⁻¹), VA999024 (20 μmol kg⁻¹) and/or VA999026 (20 μmol kg⁻¹) were injected i.p. at −30 min. Control animals were injected with saline (NaCl). All treatments were repeated twice at 2-h intervals using half of the initial dose. Values are means+s.e.mean (n=5–10). ^##P<0.01 vs controls without CRL; ^**P<0.05 vs CRL+ICAT. (c) Photomicrographs of pancreatic blood vessels at 6 h. Dashed lines delineate areas of dense extravascular erythrocyte accumulation (haematoxylin and eosin stain; scale bar: 100 μm).

Figure 2

Catalytic activities of (a) tissue kallikrein (TK) and (b) plasma kallikrein (PK), and inhibitory activities of (c) α₁-antitrypsin (α₁-AT) and (d) α₂-macroglobulin (α₂-M) in the pancreatic tissue during caerulein-induced pancreatitis. Pretreatments with the kinin B₂ receptor antagonist icatibant (ICAT; 100 nmol kg⁻¹), the TK inhibitor VA999024 (TKI; 20 μmol kg⁻¹) and/or the PK inhibitor VA999026 (PKI; 20 μmol kg⁻¹) were given i.p. 30 min before caerulein; all i.p. injections were repeated twice at half doses at 2-h intervals; control animals were injected i.p with 154 mmol L⁻¹ NaCl. Measurements were taken at 6 h. ^#P<0.05, ^##P<0.01 vs controls without CRL; ^*P<0.05, ^**P<0.01 vs CRL alone. Values are means+s.e.mean; n=5–10.

Figure 3

Effects of the tissue kallikrein (TK) inhibitor VA999024 (TKI) and the plasma kallikrein (PK) inhibitor VA999026 (PKI) in caerulein (CRL)-induced pancreatitis after treatment with the B₂ antagonist icatibant (ICAT). (a) Oedema formation and (b) haemoglobin accumulation in the pancreatic tissue: CRL or phosphate-buffered saline (PBS) was infused i.v. for 2 h. Pretreatments with icatibant (100 nmol kg⁻¹), VA999024 (20 μmol kg⁻¹) and/or VA999026 (20 μmol kg⁻¹) were given i.p. at −30 min. Control animals were injected with saline (NaCl). All treatments were repeated twice at 2-h intervals using half of the initial dose. Values are means+s.e.mean (n=5–10). ⁺P<0.05, ⁺⁺P<0.01 vs controls ICAT alone; ^#P<0.05 vs CRL+ICAT. (c) Photomicrographs of pancreatic blood vessels taken at 6 h. Dashed lines delineate areas of dense extravascular erythrocyte accumulation (haematoxylin and eosin stain; scale bar: 100 μm).

Figure 4

Catalytic activities of (a) tissue kallikrein (TK) and (b) plasma kallikrein (PK), and inhibitory activities of (c) α₁-antitrypsin (α₁-AT) and (d) α₂-macroglobulin (α₂-M) in the pancreatic tissue during caerulein-induced pancreatitis after treatment with the B₂ antagonist icatibant. The TK inhibitor VA999024 (TKI; 20 μmol kg⁻¹) and/or the PK inhibitor VA999026 (PKI; 20 μmol kg⁻¹) were given i.p. 30 min before caerulein; all i.p. injections were repeated twice at half doses at 2-h intervals; control animals were injected i.p with 154 mmol L⁻¹ NaCl. Measurements were taken at 6 h. ⁺⁺P<0.01 vs controls ICAT alone; ^#P<0.05, ^##P<0.01 vs CRL+ICAT. Values are means+s.e.mean; n=5–9.

Figure 5

Proposed mechanism of action of the kallikrein–kinin system in acute pancreatitis and differential inhibitory effects of the B₂ receptor antagonist icatibant, the selective tissue kallikrein inhibitor VA99904 and the selective plasma kallikrein inhibitor VA999026. Prevention of oedema formation by icatibant reduces plasma protein extravasation and thus attenuates the presence of endogenous kallikrein inhibitors in the pancreatic tissue, which results in pronounced augmentation of the enzymatic activity of tissue kallikrein in the pancreatic tissue. Combined treatment with the selective kallikrein inhibitors VA999024 and VA999026 also reduces plasma protein extravasation by inhibition of kinin generation, but the lack of endogenous kallikrein inhibitors in the tissue caused by this treatment is compensated by the direct inhibition of kallikreins by VA999026 and VA999026.