Glucose dialysate induces mitochondrial DNA damage in peritoneal mesothelial cells.
BACKGROUND
It is known that peritoneal mesothelial cells (PMCs) are denuded in patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD); the mechanism of damage is not well known. A high quantity of glucose loaded onto PMCs in these patients may generate toxic radicals during the mitochondrial metabolism, leading to mitochondrial DNA damage that accumulates due to the incomplete repair system of this DNA.


OBJECTIVE
To study damage to the PMCs of long-term CAPD patients, and to examine whether glucose overload accelerates this damage in vitro.


METHODS
Descriptive clinical and in vitro study.


METHODS
Stable CAPD patients and nonuremic patients undergoing elective abdominal surgery.


METHODS
(1) Clinical Samples: 13 peritoneal tissue samples from CAPD patients and 5 omental tissue samples from patients with normal renal function were investigated. PMCs in dialysate effluent were collected from another 13 stable CAPD patients. (2) In Vitro Samples: Primary PMCs were incubated for up to 144 hours in medium containing one of the following: 5.6 mmol/L glucose (control), 56 mmol/L glucose (G), 222 mmol/L glucose (high G), or 222 mmol/L mannitol (high M; osmolar control for high G). The tissues and cells of clinical and in vitro samples were stained for light and immunoelectron microscopy with anti-8-hydroxy-2'-deoxyguanosine (anti-8-OH-dG) antibody, a marker of oxidative DNA damage. In vitro cells were also studied using transmission electron microscopy. Cellular ATP content, mitochondrial membrane potential, and intracellular generation of reactive oxygen species (ROS) were analyzed by luciferase-luciferin system, or by flow cytometry using rhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA).


RESULTS
Biopsy specimens showed strong cytoplasmic staining with 8-OH-dG in patients on long-term CAPD, but only faint staining in patients with end-stage renal disease before the initiation of CAPD, and no staining in patients with normal renal function. Dialysate effluent showed strong granular staining with 8-OH-dG in most PMCs in all long-term CAPD patients, but only faint and focal staining in patients at the start and after 3-5 months of CAPD. In vitro experiments also showed strong granular staining by 8-OH-dG in most PMCs cultured in high G, weak staining in G and high M, and no staining in the control. Immunoelectron microscopy revealed the localization of 8-OH-dG to mitochondria. Transmission electron microscopy showed swelling of mitochondria, with decreased cristae, in PMCs cultured in high G. However, only partial expansion of mitochondria was seen in G and high M, and no changes were seen in the control. Cellular ATP content and mitochondrial membrane potential were reduced early, followed by an increase when cultured in high G. Intracellular ROS production was also increased in PMCs cultured in high G and high M.


CONCLUSIONS
These data suggest that high-glucose peritoneal dialysate may promote oxidative mitochondrial DNA damage in PMCs in CAPD patients.
Ishibashi
Yoshitaka
Y
Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Japan. YI431204@aol.com
Sugimoto
Tokuichiro
T
Ichikawa
Yasuko
Y
Akatsuka
Akira
A
Miyata
Toshio
T
Nangaku
Masaomi
M
Tagawa
Hitoshi
H
Kurokawa
Kiyoshi
K
D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D046508:Culture Techniques; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000187:drug effects* / Q000378:metabolism; D015314:Dialysis Solutions / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D008875:Middle Aged; D009852:Omentum / Q000187:drug effects / Q000378:metabolism; D010531:Peritoneal Dialysis, Continuous Ambulatory / Q000009:adverse effects*; D010537:Peritoneum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D004272:DNA, Mitochondrial; D015314:Dialysis Solutions; D007770:L-Lactate Dehydrogenase; D005947:Glucose