Acute effect of high glucose on long-term cell growth: a role for transient glucose increase in proximal tubule cell injury

Abstract

Although chronic exposure of renal cells to high glucose has been shown to cause cell injury, the effect of acute exposure has not been elucidated. In this study, we demonstrate that acute (10 min) exposure of human proximal tubule epithelial cells (hPTEC) to high glucose (25 mM) induces a time-dependent dual effect consisting of an early proliferation and a late apoptosis. Acute exposure of hPTEC to high glucose induced a twofold increase in DNA synthesis and cell number at 12 h. However, after 36 h, a significant decrease in cell growth is observed, followed by apoptosis. On glucose treatment, both p42/p44 mitogen-activated protein (MAP) kinases and the downstream signaling intermediate NF-kappaB were phosphorylated and translocated to the nucleus. Pretreatment of cells with MAP kinase and NF-kappaB-specific inhibitors abolished glucose-induced proliferation. However, these inhibitors were ineffective in preventing glucose-induced apoptosis. Interestingly, conditioned medium from cells exposed to high-glucose concentrations inhibited proliferation and concomitantly induced apoptosis in normal cells, suggesting that the inhibitory effect of glucose occurs through secretion of a secondary factor(s). In parallel to apoptosis, we observed an increased production of reactive oxygen species (ROS). Pretreatment of cells with the antioxidant N-acetyl cysteine reversed glucose-mediated ROS production and apoptosis, suggesting that ROS is involved in apoptosis. Our study demonstrates for the first time that a single high-glucose exposure for 10 min alone is sufficient to elicit proliferation and apoptosis in hPTEC and suggests that episodes of transient increase in glucose may contribute to cell damage leading to epithelial cell dysfunction.

Mechanism of Oxidative DNA Damage in Diabetes

“…Moreover, the effect of high glucose on ROS generation, Akt/tuberin phosphorylation, OGG1 downregulation, and 8-oxodG accumulation is markedly reduced by the antioxidant NAC. This is in agreement with a recent study showing that pretreatment of human proximal tubule epithelial cells with NAC reversed glucose-mediated ROS production (51). Collectively, our data indicate that ROS are signaling molecules responsible for Akt phosphorylation initiated by high glucose leading to tuberin phosphorylation and OGG1 protein downregulation.…”

“…This signaling cascade may play a role in oxidative stress-mediated DNA damage induced by hyperglycemia during diabetic nephropathy. Recurrent acute exposure of renal cells to high glucose during diabetes has been recently proposed to be involved in renal injury (51). Our data shed light on the molecular mechanisms implicated in these events.…”

Diabetes causes multiple genetic alterations and downregulates expression of DNA repair genes in the prostate

“…N-acetyl-L-cysteine (NAC; Sigma), acting as a reactive oxygen species (ROS) scavenger in this study, was bought from Sigma. For high glucose exposure, cells were maintained in the medium containing 25-75 mM glucose for various time points, with or without NAC pretreatment for 30 min 11,12 Western Blot Analysis Cell lysates were centrifuged at 14 000 g for 30 min at 41C. Equal amounts of cellular protein, as determined by Bradford dye-binding assay (Bio-Rad), were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45-mm (pore size) nitrocellulose.…”

“…RWPE1 cells were serum-deprived for 24 h, and incubated for 30 min at 371C with 10 mmol/l of DCFH-DA (Molecular Probes). 12 Cultures were then exposed to high glucose (25-75 mmol/l) for various time courses. Cells were harvested, and washed three times with PBS.…”

“…Cells treated identically were also subjected to analysis by a flow cytometer (Becton-Dickinson) at an excitation wave length of 485 nm and emission wave length of 530 nm. 12 …”