摘要
Objective To study apoptosis of monocytes from maintenance hemodialysis (HD) patients and to understand the relationship between cell apoptosis and mitochondrial changes. Methods Twenty patients on maintenance hemodialysis (mean age: 42±3.5 years) and 18 healthy volunteers (mean age: 38±5.0 years) were studied. Among the those patients there were 12 men and 8 women with a mean time on dialysis of 3±3.3 years, dialyzed with cuprophane or cellulose acetate membranes 2 or 3 times a week. Cell apoptosis is measured by flow cytometry (FCM) to detect phosphatidylserine (PS) expression on early apoptotic cells using fluorescein labeled Annexin V, simultaneously with dye exclusion of propidium iodide (PI), discriminated intact cells, apoptotic cells from necrotic cells. Mitochondrial transmembrane potential (△Ψm) assessment employed 3, 3' dihexyloxa￣carbocyanine iodide, DiOC 6 (3), a cationic mitochondrial probe. Swelling of mitochondria permeability transition (PT) was recorded as a decreased absorbency Department of Nephrology, Shanghai Medical University, Shanghai First People's Hospital, Shanghai 200080, China (Jin HM, Xu QJ and Ding YD) Shanghai Institute of Cell Biology, the Academy of Science of China (Wu ZJ, Hu W and Gao QR) at 540 nm by spectrophotometer. The loss of absorption induced by 5 mM Atractyloside (Atr) within 2 minutes was considered 100% of large amplitude swelling. Results Cultured monocytes undergoing spontaneous apoptosis increased at 24, 72 hours in HD patients compared with controls, 20.61%±8.89% vs.9.88%±2.06% (24h, P<0.05) and 41.49%±12.2% vs. 22.02%±3.0% (72h, P<0.05). Freshly isolated monocytes in HD patients had little apoptosis, however, reduction of △Ψm was detected, 42.50%±9.2%, which indicated the impairment of △Ψm preceded the exposure of PS. For swelling of PT, it was surprising to find that freshly isolated monocytes mitochondria in HD patients experienced overwhelming opening of PT 80%±5.6% compared with controls 5.3%±2.8% (P<0.001). Conclusions We have observed that mitochondria of freshly isolated monocytes from HD patients are experiencing the opening of PT and a drop of △Ψm, followed by the exposure of PS residues over culture time. The cause remains unclear. Our data indicate that swelling of PT and breakdown of △Ψm constitute the early events of monocytes appoptosis in HD patients, which can contribate to the imparred monocytes function.
Objective To study apoptosis of monocytes from maintenance hemodialysis (HD) patients and to understand the relationship between cell apoptosis and mitochondrial changes. Methods Twenty patients on maintenance hemodialysis (mean age: 42±3.5 years) and 18 healthy volunteers (mean age: 38±5.0 years) were studied. Among the those patients there were 12 men and 8 women with a mean time on dialysis of 3±3.3 years, dialyzed with cuprophane or cellulose acetate membranes 2 or 3 times a week. Cell apoptosis is measured by flow cytometry (FCM) to detect phosphatidylserine (PS) expression on early apoptotic cells using fluorescein labeled Annexin V, simultaneously with dye exclusion of propidium iodide (PI), discriminated intact cells, apoptotic cells from necrotic cells. Mitochondrial transmembrane potential (△Ψm) assessment employed 3, 3' dihexyloxa￣carbocyanine iodide, DiOC 6 (3), a cationic mitochondrial probe. Swelling of mitochondria permeability transition (PT) was recorded as a decreased absorbency Department of Nephrology, Shanghai Medical University, Shanghai First People's Hospital, Shanghai 200080, China (Jin HM, Xu QJ and Ding YD) Shanghai Institute of Cell Biology, the Academy of Science of China (Wu ZJ, Hu W and Gao QR) at 540 nm by spectrophotometer. The loss of absorption induced by 5 mM Atractyloside (Atr) within 2 minutes was considered 100% of large amplitude swelling. Results Cultured monocytes undergoing spontaneous apoptosis increased at 24, 72 hours in HD patients compared with controls, 20.61%±8.89% vs.9.88%±2.06% (24h, P<0.05) and 41.49%±12.2% vs. 22.02%±3.0% (72h, P<0.05). Freshly isolated monocytes in HD patients had little apoptosis, however, reduction of △Ψm was detected, 42.50%±9.2%, which indicated the impairment of △Ψm preceded the exposure of PS. For swelling of PT, it was surprising to find that freshly isolated monocytes mitochondria in HD patients experienced overwhelming opening of PT 80%±5.6% compared with controls 5.3%±2.8% (P<0.001). Conclusions We have observed that mitochondria of freshly isolated monocytes from HD patients are experiencing the opening of PT and a drop of △Ψm, followed by the exposure of PS residues over culture time. The cause remains unclear. Our data indicate that swelling of PT and breakdown of △Ψm constitute the early events of monocytes appoptosis in HD patients, which can contribate to the imparred monocytes function.
