Diabetic nephropathy is a major complication of diabetes leading to
end-stage renal disease, which requires
hemodialysis. Although the mechanism by which it progresses is largely unknown, the role of
hyperglycemia-derived oxidative stress has recently been the focus of attention as the cause of
diabetic complications. Constituent cells of the renal glomeruli have the capacity to release
reactive oxygen species (ROS) upon stimulation of
NADPH oxidase activated by
protein kinase C (PKC).
Hyperglycemia and
insulin resistance in the diabetic state are often associated with activation of PKC and
tumor necrosis factor (
TNF)-alpha, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and
TNF-alpha in rat glomeruli. Isolated rat glomeruli were stimulated with
phorbol 12-myristate 13-acetate (PMA) and
TNF-alpha, and the amount of ROS was measured using a chemiluminescence method. Stimulation with PMA (10 ng/ml) generated ROS with a peak value of 136+/-1.2 cpm/mg
protein (mean+/-SEM). The PKC inhibitor
H-7, the
NADPH oxidase inhibitor
diphenylene iodonium and the phosphatidylinositol-3 (PI-3)
kinase inhibitor
wortmannin inhibited PMA-induced ROS production by 100%, 100% and 80%, respectively. In addition,
TNF-alpha stimulated ROS production (283+/-5.8/mg
protein/20 min). The
phosphodiesterase inhibitor cilostazol activates
protein kinase A and is reported to improve
albuminuria in diabetic rats.
Cilostazol (100 microg/ml) inhibited PMA, and
TNF-alpha-induced ROS production by 78+/-1.8, and 19+/-2.7%, respectively. The effects of
cilostazol were not additive with
wortmannin.
Cilostazol arrests oxidative stress induced by PKC activation by inhibiting the
PI-3 kinase-dependent pathway, and may thus prevent the development of
diabetic nephropathy.