Functional and Morphological Change of Mitochondria in High Glucose Induced Human Peritoneal Methothelial Cells Apoptosis
Date Issued
2012
Date
2012
Author(s)
Yang, Te-Cheng
Abstract
Background and Purpose
Peritoneal dialysis (PD) is a continuous mode of dialysis, using the patient''s peritoneum as a dialysis membrane across which fluid and metabolic waste products are exchanged from the blood. However, long-term use of PD solution impact a great harm to peritoneum, resulting in human peritoneal mesothelial cells (HPMCs) apoptosis, ECM accumulation, peritoneal fibrosis and ultimately peritoneal function failure. Current conventional PD solution has many characters of bioincompatibility, such as high glucose,high osmotic pressure, and low pH value. It has been pointed out that high glucose is the major reason to cause HPMCs apoptosis. But its pathways and regulatory mechanism haven''t yet been completely clarified. Identification of mechanisms of high glucose induced HPMCs apoptosis could be useful in clinical applications to maintain the function of peritoneum for peritoneal dialysis.
Methods
HPMCs were cultured in media containing 5 (as control), 84, 138, 236 mM glucose for 24 hours. Survival rate of HPMCs was evaluated and HPMCs apoptosis was analyzed by Annexin-V/PI stain and flow cytometry. Cytochrome c release, caspase activation and PARP-1 cleavage were evaluated by Western blot. In addition, ROS generation and ensuing lipid peroxidation and oxidative damage of DNA were measured. Mitochondrial membrane potential (MMP) change was monitored by JC-1 dye. Furthermore, the antioxidant NAC is used to pretreat HPMCs before exposure to high glucose to determine whether HG-induced apoptosis of HPMCs could be contributed to ROS overproduction. Finally, mitochondrial morphology change was observed under TEM and confocal microscopy after staining by MitoTracker Red.
Results
After exposure to high glucose, the viability of HPMCs decreased and there is a significant increase in apoptosis of HPMCs. The results of Western blot assay on cell lysate showed cytochrome c release from mitochondria increased and subsequent caspase activation cascades and PARP-1 cleavage could be also demonstrated in HG condition. In addition, HG induced decreasing of MMP, assayed by JC-1. NAC exhibited attenuating effect on HG-induced ROS production, MMP decrease and HPMCs apoptosis simultaneously. Finally, under confocal microscopy and TEM, high glucose was found to cause mitochondria shortening and fragmentation.
Discussion and Prospective
In this study, we examined the correlations among high glucose, ROS, MMP, and morphological change of mitochondria, and clarified whether and how they participate in HG-induced HPMCs apoptosis. One of the initial consequences of HG is an increased generation of ROS, which is a pervasive source to injure mitochondria by causing lipid peroxidation and mtDNA damage. ROS-mediated reducing of MMP and subsequent opening of the mitochondrial permeability transition pore (mPT pore) cause cytochrome c release, and trigger the intrinsic pathway of apoptosis. In addition, we found that mitochondria undergo fragmentation concomitantly after exposure to high glucose. It may perturb structural organization of ETC connectivity, and is causally associated with ROS overpruduction and HPMCs apoptosis. Further understanding the mechanism by which HG induced HPMCs apoptosis is vital before a therapeutic intervention can be developed to maintain peritoneal membrane function for the PD patients.
Subjects
peritoneal dialysis
high glucose
human peritoneal mesothelial cells apoptosis
reactive oxygen species
mitochondria fission
Type
thesis
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