Assessment of myeloperoxidase activity in renal tissue after ischemia/reperfusion.
We have shown that a photometric assay of myeloperoxidase derived from rat blood polymorphonucleocytes employing 3,3',5,5'-tetramethylbenzidine as substrate is more sensitive than an established assay employing o-dianisidine. We went on to demonstrate that rat renal tissue is capable of inhibiting peroxidase activity. This activity approached 100% when the rat renal supernate was incubated at 60 degree C for 2 h and the assay was conducted in the presence of a 10-fold higher concentration of hydrogen peroxide (H2O2). Rat kidneys undergoing 45 min ischaemia and 1,3 and 6 h reperfusion in vivo, exhibited significant increases in myeloperoxidase activity, indicating tissue polymorphonucleocyte accumulation. Monoclonal antibodies against rat intercellular adhesion molecule 1 (ICAM-1) and CD18 of beta 2-integrins administered both 5 min before a period of 45 min renal ischaemia (20 micrograms/kg i.v.) and at the commencement of 1 h reperfusion (20 micrograms/kg i.v.) reduced renal tissue polymorphonucleocyte accumulation. However, similar treatment with the parent murine antibody immunoglobulin G1 (IgG1) and an unrelated murine antibody, IgG2a, also significantly reduced renal tissue polymorphonucleocyte accumulation. In conclusion, we demonstrate that the rat renal suppression of peroxidase activity can be overcome by a combination of heat inactivation and the provision of excess assay H2O2. In addition, the available evidence suggests that murine monoclonal antibodies against rat adhesion molecules may exert non-specific actions in our model of renal ischaemia/reperfusion in vivo.
Laight
D W
DW
Pharmacology Group, Bath University, Claverton Down, UK.
Lad
N
N
Woodward
B
B
Waterfall
J F
JF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001560:Benzidines / Q000378:metabolism; D018821:CD18 Antigens / Q000276:immunology; D002863:Chromogenic Compounds / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D007668:Kidney / Q000098:blood supply* / Q000201:enzymology*; D008297:Male; D009504:Neutrophils / Q000201:enzymology; D009195:Peroxidase / Q000097:blood / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000097:blood / Q000201:enzymology*; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D001560:Benzidines; D018821:CD18 Antigens; D002863:Chromogenic Compounds; D018799:Intercellular Adhesion Molecule-1; C021758:3,3',5,5'-tetramethylbenzidine; D006735:Horseradish Peroxidase; D009195:Peroxidase