Glucotoxicity and Diabetic Nephropathy: Investigating the role of Superoxide Dismutase and Endoplasmic Reticulum Stress
PhD Studentship funded by Kidney Research UK/Diabetes UK over 3 years (March 2006 – February 2009) for Mr Elias Katsoulieris
This research project involves the investigation of oxidative and endoplasmic reticulum stress in the development of diabetic nephropathy. A major pathologic mechanism of high glucose concentrations, associated with diabetes, in various cell types involves the generation of reactive oxygen and nitrogen species and subsequent cell damage and death. Such reactive oxygen and nitrogen species include the superoxide anion (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (OH.), each damaging cellular components in a different manner, giving rise to oxidative stress. Recently, it has been shown that endoplasmic reticulum stress, a response to cell’s inability to promote protein folding, may be caused by abnormally high glucose concentrations in pancreatic beta cells where it can lead to programmed (apoptotic) cell death.
The aims of this project are to assess the nature of damage caused by oxidative and endoplasmic reticulum stress in NRK-52E proximal tubular cells and try to overcome the toxic effects of the stresses, using interventions such as superoxide dismutase (SOD) mimetics and inhibitors of endoplasmic reticulum stress. A further aim is to elucidate the interactions between the processes of oxidative and endoplasmic reticulum stress and determine common pathways which could be potential areas for intervention.
Initial experiments established cell toxicity assays for each type of stress.
Toxicity of D-glucose (5-55 mM for 1-24 h), hydrogen peroxide (0-10 mM for 2-24 h), paraquat (a superoxide anion generator) and endoplasmic reticulum stress inducers (tunicamycin and thapsigargin) were tested using established biochemical assays for measurement of mitochondrial function (MTT) and lactate dehydrogenase release (LDH). The results allowed selection of specific doses of each of the agents above, which were used for Western blots for the detection of endoplasmic reticulum stress. Cytotoxicity assays also provided the means for detection of possible protection by SOD mimetics (EUK-134 and Tempol) and endoplasmic reticulum stress inhibitors (indirubin and kenpaullone).
Initial results have demonstrated that 25 and 35 mM of D-glucose are sufficient to promote cytotoxicity in NRK-52E cells after 24 h incubation. 1 mM hydrogen peroxide and 0.5 mM paraquat produced significant cellular dysfunction and cell death after 24 h. Tunicamycin (10 μg/mL) and thapsigargin (15μM) also produced significant cytotoxicity after 24 h. While the SOD mimetics reduced the cytotoxicity caused by hydrogen peroxide and paraquat, they had no effect in glucose-induced cytotoxicity. Indirubin (3 µM for 24 h) was able to reverse the cytotoxic actions of tunicamycin and thapsigargin. Western blots using antibodies against the recognised markers of endoplasmic reticulum stress, GRP78 and GRP94, revealed that 35 mM D-glucose for 24h induced endoplasmic reticulum stress.
These initial findings suggest that short-term exposure to high glucose levels produces endoplasmic reticulum stress in renal NRK-52E cells as well as oxidative stress and further investigations are underway to confirm these findings. Future objectives include the assessment of the cellular damage caused by high glucose, e.g. if superoxide and other reactive oxygen and nitrogen species are involved, as well as the sites of damage (DNA breaks). The role of endoplasmic reticulum stress in glucotoxicity will be investigated using specific inhibitors of endoplasmic reticulum stress (e.g. indirubin and kenpaullone). In addition, the expression and function of antioxidant enzymes will be examined under conditions experienced in diabetes.
