Mechanism of glomerular abnormality by oxidative stress in diabetic nephropathy

氧化应激在糖尿病血管并发症（包括糖尿病肾病）的发展中起重要作用。在这项研究中，我们研究了糖尿病肾小球中氧化应激是否增强以及蛋白激酶C（PKC）-β激活在调节NADPH氧化酶（活性氧（ROS）的主要来源）中的作用。抗氧化酶，如过氧化氢酶，谷胱甘肽过氧化物酶，铜锌超氧化物歧化酶的mRNA表达没有改变在肾小球链脲佐菌素（STZ）诱导的糖尿病大鼠在2和4周龄。血红素加氧酶-1（HO-1）mRNA在糖尿病大鼠肾小球中表达增强。胰岛素和维生素E治疗使糖尿病肾小球HO-1的表达正常化。免疫组化分析显示HO-1的表达增强优先在肾小球细胞如系膜细胞和上皮细胞中增加。糖尿病大鼠尿8-羟基脱氧鸟苷排泄量和肾小球免疫反应染色强度明显高于对照组。通过使用选择性PKC-β抑制剂甲磺酸红细胞介素（RBX）治疗，这些症状得到改善。糖尿病肾小球NADPH氧化酶胞浆亚基p47 phox和p67 phox表达增加。NADPH氧化酶活性在糖尿病肾小球中显著增强，RBX治疗通过阻止p47 phox和p67 phox的膜转位也得到改善。腺病毒介导的PKC-β2过表达通过刺激p47 phox和p67 phox的膜转位而增强系膜细胞中ROS的产生。提示糖尿病早期肾小球氧化应激增强，NADPH氧化酶被激活，至少部分通过PKC-β依赖的p47 phox和p67 phox膜转位。

Oxidative stress is implicated to play an important role in the development of diabetic vascular complications, including diabetic nephropathy. In this study, we examined to determine whether oxidative stress is enhanced in the diabetic glomeruli and the role of protein kinase C (PKC)-β activation in regulating the NADPH oxidase, the main source of reactive oxygen species (ROS). The mRNA expression of antioxidant enzymes such as catalase, glutathione peroxidase, and CuZn-superoxide dismutase was not altered in glomeruli of streptozotocin (STZ)-induced diabetic rat at 2 and 4 weeks of age. In contrast, the mRNA expression of heme oxygenase-l (HO-1) was enhanced in diabetic glomeruli. A treatment of insulin and vitamin E normalized the expression of HO-1 in diabetic glomeruli. Immunohistochemical analysis revealed that the enhanced expression of HO-1 is preferentially increased in glomerular cells such as mesangial cells and epithelial cells. Urinary 8-hydroxydeoxyguanosine excretion and its intense imuuno-reactive staining in the glomeruli were markedly higher in diabetic than in control rats. These were ameliorated by a treatment with a selective PKC-βinhibitor, ruboxistaurin (RBX) mesylate. The expression of p47phox and p67phox, cytosolic subunits of NADPH oxidase, was increased in diabetic glomeruli. NADPH oxidase activity was significantly enhanced in diabetic glomeruli and was also improved by RBX treatment by preventing the membranous translocation of p47phox and p67phox. Adenoviral-mediated PKC-β2 overexpression enhanced ROS generation by stimulating the membranous translocation of p47phox and p67phox in cultured mesangial cells. These results suggest that oxidative stress is enhanced in the diabetic glomeruli of early stage of diabetes, and NADPH oxidase is activated, at least in part through PKC-β-dependent p47phox and p67phox membranous translocation.