Cytotoxicity of mononuclear cells as induced by peritoneal dialysis fluids: insight into mechanisms that regulate osmotic stress-related apoptosis.
OBJECTIVE
High glucose content of peritoneal dialysis fluids (PDFs) has been shown to contribute to loss of peritoneal function during long-term peritoneal dialysis. However, hyperosmolality and hypertonicity of PDF are usually seen as similar stress events inducing osmotic stress-induced programmed cell death. In this study, we examined the impact of various osmotic agents on apoptosis induced by hyperosmolar PDFs, focusing on the mechanisms underlying the lethal effects of PDFs on peripheral blood mononuclear cells (PBMCs).


METHODS
We assessed apoptosis and necrosis by annexin V-propidium iodide (PI) labeling, and caspase-3 activity by fluorescence assay. F-actin remodeling was measured using fluorescent phalloidin labeling.


RESULTS
Hyperosmolality does not cause the cytotoxicity observed with PDF, but exposure to agents incapable of permeating cell membranes results in a significant increase in the percentage of apoptotic PBMCs by annexin V-PI labeling, which is confirmed by the increase in caspase-3 activity. Interestingly, inhibition of caspase-3 by Z-VAD-FMK did not suppress apoptosis. Extracellular hypertonicity produced polymerization of filamentous actin and cell shrinkage, which displayed similar time courses. Cell shrinkage was blocked by cytochalasin D, indicating an active role for actin cytoskeleton in hypertonicity-induced cell shrinkage. F-actin polymerization was related to an increase in intracellular ionic strength. Finally, we excluded a direct role for actin remodeling in osmotic stress-induced programmed cell death.


CONCLUSIONS
Exposure to osmolytes that cannot penetrate cell membranes results in a hypertonicity-induced apoptosis that cannot be blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. In addition, extracellular hypertonicity induced by impermeant solutes produces F-actin polymerization through an increase in intracellular ionic strength. The remodeling of the cytoskeleton does not modulate apoptosis but participates in cell shrinkage.
Gastaldello
Karine
K
Laboratoire de Recherche en Nephrologie, Brussels, Belgium. kgastald@ulb.ac.be
Husson
Cecile
C
Dondeyne
Jean-Paul
JP
Vanherweghem
Jean-Louis
JL
Tielemans
Christian
C
D000199:Actins / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton; D015314:Dialysis Solutions / Q000737:chemistry* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009997:Osmotic Pressure; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation; D015398:Signal Transduction
D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D015853:Cysteine Proteinase Inhibitors; D015314:Dialysis Solutions; D006982:Hypertonic Solutions; D019384:Nucleic Acid Synthesis Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D015638:Cytochalasin D; D053148:Caspase 3