Background: Protein factors accumulating in sera of patients with end-stage renal disease (ESRD) that interfere with the nonspecific immune response by inhibiting essential functions of polymorphonuclear leucocytes (PMNLs) have previously been described. No such factor has been isolated from acute renal failure (ARF) patients to date.
Materials and methods: Using a three-step chromatographic procedure involving ion exchange, size exclusion and hydrophobic interaction chromatography we purified the apo- and holo-form of retinol binding protein (RBP) from high-flux dialyser (polyacrylonitrile; AN69) ultrafiltrates of patients with ARF. Their effect on the chemotaxis of PMNLs isolated from healthy donors was determined by the under-agarose method. Whole-blood assays applying flow cytometry were used to assess phagocytosis and the oxidative metabolism of PMNLs. Apoptosis was assessed by determining the DNA content using propidium iodide.
Results: Isolated apo- and holo-forms of RBP were truncated on their C-terminus as determined by mass spectrometry. All isolates significantly inhibited the chemotactic movement of PMNLs obtained from healthy donors and the PMNL oxidative metabolism stimulated by E. coli. These effects were concentration dependent. Retinol binding protein had no influence on the PMNL oxidative metabolism stimulated by PMA and on PMNL phagocytosis. Commercially available RBP isolated from urine influenced PMNL functions in the same way. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 significantly attenuated the phagocytosis-induced respiratory burst and RBP did not lead to a further decrease. Polymorphonuclear leucocyte apoptosis was significantly inhibited by RBP.
Conclusions: The apo- and holo-forms of RBP isolated from the ultrafiltrate of ARF patients inhibit PMNL chemotaxis, oxidative metabolism and apoptosis. Therefore, RBP may be considered a uraemic toxin contributing to a disturbed immune defence.