Related Experiment Videos
Necrosis and apoptosis of polymorphonuclear cells exposed to peritoneal dialysis fluids in vitro
M Cendoroglo1, S Sundaram, C Groves
1Department of Medicine, New England Medical Center, Boston, Massachusetts, USA.
Abstract:
Conventional peritoneal dialysis (PD) fluids are known to inhibit polymorphonuclear cells (PMN) phagocytosis, oxidative burst and enzyme release. However, the relative contributions of apoptosis and/or necrosis to this dysfunction have not been examined. We investigated the effects of osmolality, glucose concentration and heat-sterilization of PD fluids on necrosis and apoptosis of PMN. Polymorphonuclear cells were isolated from 8 healthy volunteers and exposed to different PD fluids for four hours. PMN were then double-stained with Hoechst 33342 and propidium iodide to study the proportion of viable, apoptotic and necrotic cells. Transmission electron microscopy (TEM) was performed to confirm the results obtained with flow cytometry. The fluids studied were conventionally heat-sterilized 1.5% Dianeal (1.5% D), conventionally heat-sterilized 4.25% Dianeal (4.25% D), 1.5% D in which the osmolality was increased to that of 4.25% D by adding mannitol (1.5% D + M), a filter-sterilized version of 4.25% D (4.25% D-F) and a 1.1% amino acid PD fluid (AA) (Nutrineal PD4). All PD fluids had their pH equilibrated (pH = 7.4) by the addition of sodium bicarbonate. Compared to PMN exposed to culture medium, a significantly higher proportion of necrosis was observed in PMN exposed to 1.5% D (P = 0.04). The 4.25% D induced greater necrosis than 1.5% D (P = 0.001), and the 4.25% D also induced significantly more necrosis (P = 0.002) compared to 4.25% D-F. These data suggest that the consequences of heat-sterilization, rather than high glucose concentration are responsible for the necrosis observed. Indeed, the proportion of necrotic PMN with 4.25% D-F was not significantly different from 1.5% D. The 1.5% D + M and AA induced significantly more apoptosis compared to 1.5% D (P = 0.006 and P < 0.05, respectively), suggesting that apoptosis can be induced by the high osmolality of PD fluids. However, 1.5% D +/- M also induced significantly more apoptosis (P = 0.007) compared to 4.25% D-F. This suggests that the apoptosis effect is specific for the osmolyte present in PD fluids, and that mannitol and amino acids induce more apoptosis than glucose. In summary, the different non-physiological components of conventional PD fluids evaluated in this study had a differential effect on PMN survival. Heat sterilization of high glucose-containing PD fluids was associated predominantly with necrosis of PMN, and high osmolality with apoptosis.
Insights
Heat sterilization of peritoneal dialysis fluids causes polymorphonuclear cell (PMN) necrosis, while high osmolality induces apoptosis. These findings highlight how PD fluid components affect immune cell function.
Area of Science:
- Nephrology
- Immunology
- Cell Biology
Background:
- Conventional peritoneal dialysis (PD) fluids can impair polymorphonuclear cell (PMN) function.
- The specific mechanisms, such as apoptosis or necrosis, by which PD fluids affect PMN viability are not well understood.
Purpose of the Study:
- To investigate the impact of osmolality, glucose concentration, and heat sterilization in PD fluids on PMN necrosis and apoptosis.
- To differentiate the effects of heat sterilization versus high glucose on PMN cell death.
Main Methods:
- PMN were isolated from healthy volunteers and exposed to various PD fluids (heat-sterilized and filter-sterilized Dianeal, Dianeal with added mannitol, and amino acid solution).
- Cell viability, apoptosis, and necrosis were assessed using Hoechst 33342 and propidium iodide staining, with confirmation by transmission electron microscopy (TEM).
Main Results:
- Heat-sterilized PD fluids, particularly those with high glucose (4.25% Dianeal), induced significantly more PMN necrosis compared to filter-sterilized fluids.
- High osmolality, achieved by adding mannitol or using amino acid solutions, led to a significant increase in PMN apoptosis compared to standard PD fluids.
- The apoptosis-inducing effect was specific to the osmolyte, with mannitol and amino acids showing a greater effect than glucose.
Conclusions:
- Heat sterilization of PD fluids is a primary driver of PMN necrosis, rather than high glucose concentration alone.
- High osmolality in PD fluids contributes to PMN apoptosis, with specific osmolytes like mannitol and amino acids having a more pronounced effect than glucose.
- Different components of PD fluids differentially impact PMN survival, influencing immune function during peritoneal dialysis.