Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-α stimulation, and effects of a phosphodiesterase inhibitor

Abstract

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)-α, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and TNF-α in rat glomeruli. Isolated rat glomeruli were stimulated with phorbol 12-myristate 13-acetate (PMA) and TNF-α, 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-α 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 μg/ml) inhibited PMA, and TNF-α-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.

Cilostazol Ameliorates Nephropathy in Type 1 Diabetic Rats Involving Improvement in Oxidative Stress and Regulation of TGF-β and NF-κB

“…33) Elevation of intracellular DAG levels activates protein kinase C (PKC), and subsequently activates NADPH oxidase to induce ROS. 34) A multitude of in vivo studies have been performed utilizing antioxidants in experimental diabetic models. The effects of antioxidants on oxidative stress are measured through certain observable biomarkers.…”

“…Cilostazol therapy significantly reduced the MDA level and restored the catalase and GSH levels. In 2007, Koike et al 34) found that cilostazol inhibits glomerular ROS production stimulated by phorbol 12-myristate 13-acetate (PMA) in a dose-dependent manner. Their results indicated that cilostazol inhibits the ROS generation induced by PKC activation by inhibiting the PI-3 kinase-dependent pathway in isolated rat glomeruli.…”

The Role of Inflammatory Cytokines in Diabetic Nephropathy

“…84 Recent experimental studies using isolated rat glomeruli demonstrate this cytokine activates NADPH oxidase through protein kinase C/phosphatidylinositol-3 kinase and mitogenactivated protein kinase pathways. 85 Therefore, TNF-␣, independent of hemodynamic factors, prompts the local generation of ROS, resulting in alterations of the barrier function of the glomerular capillary wall and leading to enhanced albumin permeability. 86 Experimental studies have consistently reported that mRNA encoding TNF-␣ and protein levels increase in glomerular and proximal tubule cells from diabetic rats.…”

Role of free radicals in liver diseases

“…These cells, when activated, produce and secrete cytokines; cytokines may be proinflammatory, such as TNF-a and IL-1, or profibrotic, such as TGF-b. These proteins can further increase ROS and play important roles in the mediation of hepatic injury [23,[85][86][87], such as fatty liver, by inhibiting lipase of lipoprotein and adiponectin and fibrosis as a result of HSC activation [88][89][90].…”