AGEs induce oxidative stress and activate protein kinase C-beta(II) in neonatal mesangial cells

Increased activation of specific protein kinase C (PKC) isoforms and increa sed nonenzy matic glycation of intracellular and extracellular proteins [th e accumulation of advanced glycation end products (AGEs)] are major mechani stic pathways implicated in the pathogenesis of diabetic complications. Blo cking PKC-beta(II) has been shown to decrease albuminuria in animal models of diabetes. To demonstrate a direct relationship between AGEs and the indu ction and translocation of PKC-beta(II), studies were carried out in rat ne onatal mesangial cells, known to express PKC-beta(II) in association with r apid proliferation in post-natal development. Oxidative stress was studied by using the fluorescent probe dichlorfluorescein diacetate. Translocation of PKC-beta(II) was demonstrated by using immunofluorescence and Western bl otting of fractionated mesangial cells. Induction of intracellular oxidativ e stress, increase in intracellular calcium, and cytosol to membrane PKC-be ta(II) translocation (with no change in PKC-alpha) were demonstrated after exposure to AGE-rich proteins. These data support the hypothesis that AGEs cause mesangial oxidative stress and alterations in PKC-beta(II), changes t hat may ultimately contribute to phenotypic abnormalities associated with d iabetic nephropathy.