Influence of Uremia on Cell Viability and Cytokine Release of Human Peritoneal Mesothelial Cells

Abstract

Introduction: There is still no evidence whether human peritoneal mesothelial cells (HPMC) from patients with end-stage renal failure are altered in cell viability or show a different pattern of the release of proinflammatory cytokines. Also the serum of patients with uremia may contain substances stimulating the cytokine release of HPMC. Study Design: The IL-1β-induced IL-6/IL-8 release of HPMC from healthy donors and from patients with end-stage renal disease (ESRD) were measured before the start of chronic peritoneal dialysis (PD) and during PD therapy. Additionally the influence of uremic and non-uremic serum on IL-6 and IL-8 release of normal HPMC was studied. Cell viability was assessed by MTT assay and by the measurement of intracellular ATP (chemoluminescence assay). HPMC were obtained from the following patient groups: (1) non-uremic control patients (n = 7); (2) patients with ESRD undergoing PD catheter implantation for the first time (n = 7), and (3) patients on PD undergoing catheter exchange for noninfectious reasons (n = 6). Pooled human serum from PD patients and normal controls were used for stimulation experiments. HPMC from different donors were grown to confluence (second passage) and then stimulated with IL-1β (1,000 pg/ml in M199) for 24 h. IL-6 and IL-8 concentrations were measured in the supernatant by ELISA. Additionally uremic and non-uremic sera were incubated with HPMC from normal donors for 24 h with a subsequent 24-hour IL-1β stimulation. Mesothelial cell protein mass was determined by the Bradford reagent. Results: Non-uremic patients and ESRD patients did not differ with regard to the global cell viability of HPMC according to MTT assay activity or the intracellular ATP concentration. However, HPMC from uremic patients produced more IL-8 on IL-1β stimulation than the non-uremic controls (group 2, 53.5 ± 15.7 pg/µg; group 3, 70.5 ± 27.3 pg/µg vs. group 1, 24.0 ± 11.8 pg/µg). HPMC from patients on chronic PD additionally released significantly more IL-6 (30.5 ± 13.8 pg/µg) on IL-1β stimulation than uremic patients before the onset of PD (6.2 ± 2.6 pg/µg; p < 0.01). Incubation of normal HPMC with the serum from uremic donors produced an enhanced stimulated IL-8 release compared to the exposition with normal control serum (50.6 ± 6.1 vs. 20.8 ± 2.9 pg/µg; p < 0.01). Conclusion: HPMC from uremic patients more readily release IL-8 on stimulation with IL-1β. On chronic PD treatment IL-6 release was further enhanced. Not further classified serum components in uremia also enhance IL-6 and IL-8 release of HPMC.