St important mediators released by the endothelium is NO. NO acts as a potent vasodilator, and also inhibits inflammation, growth of vascular smooth muscle and aggregation of platelets [20?3]. Dysregulation of NO has been described in patients with DN, including increased NO expression in early DN, followed by marked down-regulation. Henke et al. [24] generated mice in which the nuclear AZ 876 site factor kappa B (NF-kB) suppressor IkBaD was induced in the endothelium using Cre/Lox technology. When these mice were exposed to Angiotensin II infusion, high salt and inhibition of endogenous NO production, hypertension was not prevented. However, NF-kB suppression markedly reduced renal injury as evidenced by decreased proteinuria, renal inflammation and fibrosis [24]. This study demonstrated a previously unappreciated role of the endothelium in ML 264 site glomerular injury [25]. It is believed that the glomerular filtration barrier (GFB), including the podocyte layer, the glomerular basement membrane (GBM), and the endothelium, plays an essential role in regulating glomerular permeability. Recent studies have 
demonstrated the importance of the glomerular endothelium and its surface layer inGlomerular Endothelial Cell InjuryFigure 1. Pathological characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. PAS staining of sections from NS (A C) and ADR-injected (B D) wild type (A B) and eNOS-deficient (C D) mice at day 28. Masson trichrome staining of sections from NS (E G) and ADR-injected (F H) wild type (E F) and eNOS-deficient (G H) mice at day 28. eNOS-deficient mice with ADR-induced nephropathy exhibited well developed exudative (fibrin-cap) lesions, glomerular sclerosis, interstitial fibrosis and inflammation at day 28. Original magnifications, 400 X. doi:10.1371/journal.pone.0055027.gpreventing proteinuria [26]. Increasing evidence also demonstrates that glomerular endothelial cell fenestrae are integral components of the glomerular filtration barrier [27?1]. Reduction in glomerular endothelial cell fenestration and an increase in podocyte detachment are correlated with the severity of classical DN lesions and renal function in type 1 diabetic patients [32]. Taken together, these studies from both structural and functional viewpoints demonstrate that glomerular endothelial dysfunction plays a critical role in the pathogenesis of progressive renal disease, suggesting that endothelial function is also a key determinant of susceptibility to nephropathy. In the present study we hypothesize that endothelial dysfunction can initiate and propel the development and progression of glomerulopathy. We tested whether eNOS deficiency promotes endothelial injury and drives the development of adriamycin (ADR)-induced nephropathy in C57BL/6 mice, an ADR-resistant strain. We 1527786 also examined and compared podocyte and glomerular endothelial cell injury in ADR-induced nephropathy in Balb/c mice, an ADR-susceptible strain. Finally we investigated whether the conditioned medium from mouse microvascular endothelial cells over expressing eNOS can protect podocytes from TNF-ainduced injury in vitro.which adheres to the “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.” Five C57BL/6 male mice and six Balb/c male mice per group were used in each experiment. To establish the animal model of adriamycin (ADR)-induced nephropathy, wild type, eNOS knockout and Balb/c mice received a single intravenous injection of ADR (10.5 mg/kg;.St important mediators released by the endothelium is NO. NO acts as a potent vasodilator, and also inhibits inflammation, growth of vascular smooth muscle and aggregation of platelets [20?3]. Dysregulation of NO has been described in patients with DN, including increased NO expression in early DN, followed by marked down-regulation. Henke et al. [24] generated mice in which the nuclear factor kappa B (NF-kB) suppressor IkBaD was induced in the endothelium using Cre/Lox technology. When these mice were exposed to Angiotensin II infusion, high salt and inhibition of endogenous NO production, hypertension was not prevented. However, NF-kB suppression markedly reduced renal injury as evidenced by decreased proteinuria, renal inflammation and fibrosis [24]. This study demonstrated a previously unappreciated role of the endothelium in glomerular injury [25]. It is believed that the glomerular filtration barrier (GFB), including the podocyte layer, the glomerular basement membrane (GBM), and the endothelium, plays an essential role in regulating glomerular permeability. Recent studies have demonstrated the importance of the glomerular endothelium and its surface layer inGlomerular Endothelial Cell InjuryFigure 1. Pathological characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. PAS staining of sections from NS (A C) and ADR-injected (B D) wild type (A B) and eNOS-deficient (C D) mice at day 28. Masson trichrome staining of sections from NS (E G) and ADR-injected (F H) wild type (E F) and eNOS-deficient (G H) mice at day 28. eNOS-deficient mice with ADR-induced nephropathy exhibited well developed exudative (fibrin-cap) lesions, glomerular sclerosis, interstitial fibrosis and inflammation at day 28. Original magnifications, 400 X. doi:10.1371/journal.pone.0055027.gpreventing proteinuria [26]. Increasing evidence also demonstrates that glomerular endothelial cell fenestrae are integral components of the glomerular filtration barrier [27?1]. Reduction in glomerular endothelial cell fenestration and an increase in podocyte detachment are correlated with the severity of classical DN lesions and renal function in type 1 diabetic patients [32]. Taken together, these studies from both structural and functional viewpoints demonstrate that glomerular endothelial dysfunction plays a critical role in the pathogenesis of progressive renal disease, suggesting that endothelial function is also a key determinant of susceptibility to nephropathy. In the present study we hypothesize that endothelial dysfunction can initiate and propel the development and progression of glomerulopathy. We tested whether eNOS deficiency promotes endothelial injury and drives the 
development of adriamycin (ADR)-induced nephropathy in C57BL/6 mice, an ADR-resistant strain. We 1527786 also examined and compared podocyte and glomerular endothelial cell injury in ADR-induced nephropathy in Balb/c mice, an ADR-susceptible strain. Finally we investigated whether the conditioned medium from mouse microvascular endothelial cells over expressing eNOS can protect podocytes from TNF-ainduced injury in vitro.which adheres to the “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.” Five C57BL/6 male mice and six Balb/c male mice per group were used in each experiment. To establish the animal model of adriamycin (ADR)-induced nephropathy, wild type, eNOS knockout and Balb/c mice received a single intravenous injection of ADR (10.5 mg/kg;.