High Glucose-Induced Oxidative Stress Increases the Copy Number of Mitochondrial DNA in Human Mesangial Cells

Abstract

Oxidative damage to mitochondrial DNA (mtDNA) has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS) to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP) on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells cultured in 5 mM glucose, 25 mM glucose, and mannitol (osmotic control), as well as in cells cultured in 25 mM glucose in the presence and absence of 200 μM MnTBAP. Intracellular ROS was assessed by confocal microscopy and flow cytometry in human mesangial cells. The copy number of mtDNA was significantly increased when human mesangial cells were incubated with 25 mM glucose compared to 5 mM glucose and mannitol. In addition, 25 mM glucose rapidly generated ROS in the cells, which was not detected in 5 mM glucose. Furthermore, mtDNA copy number was significantly decreased and maintained to normal following treatment of cells with 25 mM glucose and MnTBAP compared to 25 mM glucose alone. Inclusion of MnTBAP during 25 mM glucose incubation inhibited mitochondrial superoxide in human mesangial cells. Increased mtDNA copy number in human mesangial cells by high glucose could contribute to increased mitochondrial superoxide, and prevention of mtDNA copy number could have potential in retarding the development of diabetic nephropathy.

Time‐course effect of high‐glucose‐induced reactive oxygen species on mitochondrial biogenesis and function in human renal mesangial cells

“…Similar beneficial effect of MnTBAP in preventing the copy number of mtDNA in high-glucose-cultured renal mesangial cells was observed in our previous study (Al-Kafaji and Golbahar, 2013), supporting a significant role of MnSOD, a primary scavenger of mitochondrial superoxide radicals, in protecting the mitochondria from oxidative stress. Similar beneficial effect of MnTBAP in preventing the copy number of mtDNA in high-glucose-cultured renal mesangial cells was observed in our previous study (Al-Kafaji and Golbahar, 2013), supporting a significant role of MnSOD, a primary scavenger of mitochondrial superoxide radicals, in protecting the mitochondria from oxidative stress.…”

“…MitoSOX red dye (Invitrogen) was used to detect the production of mitochondrial superoxide in human renal mesangial cells as previously described (Al-Kafaji and Golbahar, 2013). MitoSOX is a fluorogenic dye, which permeates live cells where it selectively targets mitochondria and readily oxidized by superoxide.…”

“…Genomic DNA was extracted from human renal mesangial cells using the QIAamp DNA Mini Kit (Qiagen) as previously described (Al-Kafaji and Golbahar, 2013). Purity and concentration of DNA recovered were determined with a Nano Drop spectrophotometer.…”

Diabetic nephropathy in type 1 diabetes: a review of early natural history, pathogenesis, and diagnosis

“…In addition, overproduction of ROS by high glucose concentrations compromises the antioxidant defense mechanisms in DN such as reduced levels of mitochondrial‐specific manganese superoxide dismutase that further aggravates oxidative stress. Oxidative stress including ROS may damage mitochondrial DNA and impair electron transport chain, leading to increased ROS production Accumulation of AGEs.…”

“…Oxidative stress including ROS may damage mitochondrial DNA and impair electron transport chain, leading to increased ROS production. 32 • Accumulation of AGEs. Advanced glycation that occurs at an accelerated rate in T1D is a prominent phenomenon in the kidney.…”