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

Abstract

The present study investigated the time-course effect of high-glucose-induced reactive oxygen species (ROS) on mitochondrial biogenesis and function in human renal mesangial cells and the effect of direct inhibition of ROS on mitochondria. The cells were cultured for 1, 4, and 7 days in normal glucose or high glucose in the presence and absence of Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) or catalase. Mitochondrial ROS production was assessed by confocal microscope. mtDNA copy number and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factors 1 (NRF-1), and mitochondrial transcription factor A (TFAM) transcripts were analyzed by real-time PCR. PGC-1α, NRF-1, and TFAM proteins were analyzed by Western blotting. Mitochondrial function was determined by assessing mitochondrial membrane potential and adenosine triphosphate (ATP) levels. High glucose induced significant increases in mitochondrial superoxide and hydrogen peroxide (H2 O2 ) at day 1, which remained significantly elevated at days 4 and 7. The copy number of mtDNA and expression of PGC-1α, NRF-1, and TFAM were significantly increased at 1 day in high glucose but were significantly decreased at 4 and 7 days. A progressive decrease in mitochondrial membrane potential was observed at 1, 4, and 7 days in high glucose, and this was associated with decreased ATP levels. Treatment of cells with MnTBAP or catalase during high-glucose incubation attenuated ROS production and reversed the alterations in mitochondrial biogenesis and function. Increased mitochondrial biogenesis in human renal mesangial cells may be an early adaptive response to high-glucose-induced ROS, and prolonged ROS production induced by chronic high glucose decreased mitochondrial biogenesis and impaired mitochondrial function. Protection of mitochondria from high-glucose-induced ROS may provide a potential approach to retard the development and progression of diabetic nephropathy.

The epigenetic landscape related to reactive oxygen species formation in the cardiovascular system

“…Redox agents such as H 2 S decreased TFAM promoter methylation, most likely by inhibiting DNMT3A expression, thereby maintaining mtDNA copy numbers in vascular cells (Li and Yang, ). Several other studies have reported that ROS can elevate mtDNA copy numbers (Lee and Wei, ; Hori et al , ; Marine et al , ), although this effect may be time‐ and dose‐dependent (Al‐Kafaji et al , ) and possibly also dependent on the type of redox modulation. For example, in mice exposed to cigarette smoke extracts, TFAM promoter methylation was increased resulting in strongly reduced TFAM expression and endothelial cell apoptosis (Zhang et al , ).…”

Peripheral blood mitochondrial DNA copy number as a novel potential biomarker for diabetic nephropathy in type�2 diabetes patients

“…qPCR was performed with a 7900HT real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following cycling conditions: 1 cycle of 50˚C for 2 min, 1 cycle of 95˚C for 10 min, and 30 cycles of 95˚C for 15 sec and 60˚C for 1 min. For each PCR run, standard curves were established from 5 serially diluted, purified PCR products (1x10 2 to 1x10 7 ng/ml) from the CYTB gene and the B2M gene to generate quantification data as previously described (19,21). Quantification of mtDNA was accomplished by calculating the ratio of mtDNA-encoded CYTB gene to the nDNA-encoded B2M gene and mtDNA-CN data were expressed as mtDNA per nDNA.…”

“…Compared to nuclear DNA (nDNA), mtDNA is more susceptible to oxidative stress due to its close proximity to the site of free radical generation (17). Increased oxidative stress in human cells may contribute to alterations in the mtDNA-CN, mtDNA-encoded gene expression and mitochondrial abundance (18)(19)(20)(21). A previous study by our group has indicated that in response to high glucose-induced chronic oxidative stress, the renal mtDNA-CN is decreased and the mitochondria are impaired (21).…”

“…Increased oxidative stress in human cells may contribute to alterations in the mtDNA-CN, mtDNA-encoded gene expression and mitochondrial abundance (18)(19)(20)(21). A previous study by our group has indicated that in response to high glucose-induced chronic oxidative stress, the renal mtDNA-CN is decreased and the mitochondria are impaired (21). Furthermore, decreased mtDNA-CN in the peripheral blood has also been reported in several diseases associated with oxidative stress.…”

Nox4 is involved in high glucose-induced apoptosis in renal tubular epithelial cells via Notch pathway

“…Reactive oxygen species (ROS) have been revealed as important signaling molecules that mediate renal injury in patients with diabetes (1). High glucose (HG) increases intracellular ROS levels in renal cells and contributes to the development and progression of diabetic renal injury (2,3). Under normal physiological conditions, low levels of ROS are produced by the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase (Nox) family as byproducts of the mitochondrial electron transport chain, and are important in the regulation of various cellular functions including inflammatory gene expression, proliferation and apoptosis (4).…”