The uraemic retention solute para-hydroxy-hippuric acid attenuates apoptosis of polymorphonuclear leukocytes from healthy subjects but not from haemodialysis patients

Abstract

BACKGROUND: Disturbed polymorphonuclear leukocyte (PMNL) apoptosis contributes to the dysregulation of the non-specific immune system in uraemia. Intracellular Ca(2+) modulates PMNL apoptotic cell death. We investigated the effect of para-hydroxy-hippuric acid (PHA), an erythrocyte plasma membrane Ca(2+)-ATPase inhibitor accumulating in uraemic sera, and of cyclopiazonic acid (CPA), an inhibitor of the sarko/endoplasmatic Ca(2+)-ATPase, on PMNL apoptosis. METHODS: Apoptosis of PMNLs from healthy subjects and from haemodialysis (HD) patients was assessed after incubation for 20 h by evaluating morphological features under the fluorescence microscope and by measuring the DNA content and caspase activities by flow cytometry. The intracellular calcium concentration ([Ca(2+)](i)) was determined by measurement of fura-2 fluorescence using the 340/ 380 nm dual wavelength excitation. RESULTS: Spontaneous apoptosis of PMNLs from healthy subjects and from HD patients did not differ. PHA significantly attenuated, while CPA increased, the apoptotic cell death of PMNLs from healthy subjects. The PHA effect was not observed with PMNLs from HD patients, irrespective of whether the blood was drawn before or after HD treatment. Baseline [Ca(2+)](i) was increased in PMNLs obtained from HD patients before dialysis but reversed after dialysis. The PHA effects were not mediated via [Ca(2+)](i). The chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induced a [Ca(2+)](i) increase and reduced PMNL survival. Extracellular Ca(2+) did not affect CPA- and fMLP-induced apoptosis. CONCLUSIONS: PHA, without affecting [Ca(2+)](i), attenuates apoptosis of healthy but not of uraemic PMNLs. CPA and fMLP enhance PMNL apoptosis independently of Ca(2+) influx.