Caspase-3-dependent pathway mediates apoptosis of human mononuclear cells induced by cellulosic haemodialysis membranes.
BACKGROUND
Mononuclear cells from patients dialysed with cellulosic membranes undergo rapid apoptosis in vitro. The aim of the present study was to determine whether the apoptosis associated with cellulosic haemodialysis membrane shares similar features with the spontaneous apoptosis described in normal monocytes. Thus, we determined whether apoptosis is dependent on caspase-3 activity and is inhibited by lipopolysaccharide (LPS), which are two features of spontaneous apoptosis in normal monocytes.


METHODS
We examined mononuclear cells from healthy subjects and from 14 end-stage renal failure patients on haemodialysis with cellulosic membranes (n=7) and non-cellulosic membranes (n=7). Isolated mononuclear cells were cultured for 48 h. To determine the effect of haemodialysis membrane exposure on caspase-3 activity, on mononuclear apoptosis, or both, cells from healthy subjects were cultured in mini-dialysers with the same membrane types that were used in the haemodialysis patients. Caspase-3 active form was determined by flow cytometric analysis using anti-human-active caspase-3 antibodies. The effect of LPS and Ac-DEVD-CHO, a specific inhibitor of active caspase-3, was also evaluated. Cell apoptosis was assessed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling method.


RESULTS
After 48 h of culture, the percentage of mononuclear cells expressing the active form of caspase-3 was greater in patients dialysed with cellulosic membranes than in patients using non-cellulosic membranes and in healthy subjects. This increase in caspase-3 activity was associated with a high rate of apoptosis, which was prevented by Ac-DEVD-CHO, an inhibitor of caspase-3 activity. LPS decreased both apoptosis and caspase-3 activity in mononuclear cells from patients dialysed with cellulosic membranes. Finally, in cells from healthy subjects, both caspase-3 activation and apoptosis were induced after incubation with cellulosic membranes. In contrast, the active form of caspase-3 was not increased in cells cultured with non-cellulosic membranes and was significantly lower than with cellulosic membranes.


CONCLUSIONS
These findings suggest that the apoptosis of mononuclear cells induced by cellulosic haemodialysis membranes occurs through a pathway that is similar to the spontaneous apoptosis of normal monocytes. They additionally suggest that LPS regulates the proteolytic activation of caspase-3.
Carracedo
Julia
J
Unidad de Investigación, Servicio de Nefrología, Hospital Universitario Reina Sofía, Cordoba, Spain.
Ramírez
Rafael
R
Soriano
Sagrario
S
Martín-Malo
Alejandro
A
Rodríguez
Mariano
M
Aljama
Pedro
P
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D002482:Cellulose; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008567:Membranes, Artificial; D008875:Middle Aged; D009000:Monocytes / Q000502:physiology*; D006435:Renal Dialysis
D008070:Lipopolysaccharides; D008567:Membranes, Artificial; D002482:Cellulose; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases