Avaliação in vitro do P-cresol sobre o metabolismo oxidativo e apoptose dos neutrófilos de cães

Abstract

In humans, the immunosuppression observed in uremic patients is associated with the neutrophil dysfunction caused by uremic toxins. P-cresol accumulates in the blood of patients with chronic renal failure (CRF) promoting the inhibition of neutrophil oxidative metabolism. This study aimed to measure the plasma concentrations of p-cresol in dogs with CRF and test the hypothesis that free p-cresol causes neutrophil dysfunction. To this end, plasma concentration of p-cresol was determined using high pressure liquid chromatography (HPLC) in dogs presenting stage IV of CRF. The in vitro specific effect of p-cresol on the oxidative metabolism and apoptosis rate of neutrophils from healthy dogs was also evaluated, considering the highest plasma concentration of this toxin observed on the in vivo measurement of dogs with CRF. Isolated neutrophils from 20 healthy dogs were incubated in RPMI medium alone, supplemented with p-cresol or plasma from dogs with CRF. Neutrophil oxidative metabolism was assessed by capillary flow cytometry using the probes hydroethidine and 2',7'- dichlorofluorescein diacetate with or without stimulus with phorbol myristate acetate and N-formyl-methionyl-leucylphenylalanine. Apoptosis and viability of neutrophils were quantified also in capillary flow cytometry using Annexin V-PE system with or without stimulus with camptothecin. Dogs with CRF presented oxidative stress and increased plasma levels of p-cresol, the higher concentration of p-cresol caused in vitro neutrophil dysfunction. It can be concluded that p-cresol may be involved with in vivo oxidative stress that its higher concentration decreases the viability and lead to a reduced production of reactive oxygen species