ISH NIA PS 01-02 Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

Abstract

Objective: Increased generation of reactive oxygen species (ROS) is a common pathogenic mechanism underlying vascular and renal complications in diabetes. Endothelial NAD(P)H oxidase is a major source of vascular ROS and plays important role in endothelial dysfunction. We hypothesize that activation of endothelial NAD(P)H oxidase would initiate and accelerate diabetic nephropathy, especially development of albuminuria, in diabetic milieu. Design and Method: We used endothelial specific NOX2 transgenic (Es-NOXTg) mice, AKITA type1 diabetic (AKITA) mice, NOX2Tg crossbred with AKITA mice, and wild type (WT) mice. Es- NOXTg was generated in which NOX2, gp91 phox of NAD(P)H oxidase, under the control of Tie2 promoter, was overexpressed in the endothelium. All mice were back-crossed into C57BL/6J strain. These mice were sacrificed at 6 and 12-week-old of ages for molecular and histological analysis. We applied the in vivo live imaging techniques with multi-photon laser microscopy and various sizes of FITC labeled dextrans to analyze alterations in permeability of glomerular capillary walls in disease conditions. Results: Urinary albumin excretion was increased only in NOXTG-AKITA mice but not in WT and AKITA mice at 6-week-old. At 12-weeks-old, serum creatinine level was significantly elevated only in NOXTg-AKITA but not AKITA and WT mice. No significant morphological changes were detected in glomeruli from all groups by light microscopic examinations. But slight degree of structural changes in podocytes and mesangial cells were observed only in NOXTg-AKITA mice under the electron microscope. The in vivo live imaging techniques revealed increased filtration of 40kDa dextran in glomeruli in AKITA and NOXTg-AKITA, but not in WT mice. Moreover, increased permeability of larger molecules, 70 KDa dextran, were detected in NOXTg-AKITA mice. Lectin (WGA lectin) staining was decreased along glomerular endothelium in NOXTg-AKITA mice. Conclusions: Activation of endothelial NAD(P)H oxidase in hyperglycemic milieu initiated and accelerated diabetic nephropathy characterized by development of albuminuria and hyperfiltration of macromolecules.