For the experimental study, human mesangial cells were incubated in DMEM with normal glucose (NG; 5.5 mM) and high blood sugar (HG; 25 mM) for 24 hours after serum starvation. therapeutic target for diabetic nephropathy. Introduction Diabetic nephropathy is usually a leading cause of end-stage kidney disease (ESKD) in developed countries. In earlier stage, glomerular hyperfiltration, glomerular hypertrophy, glomerular Cloxiquine basement membrane (GBM) thickening, and microalbuminuria are generally observed, followed by mesangial matrix growth and proteinuria. Subsequently, nodular glomerulosclerosis and massive proteinuria develop in the advanced stage, leading to ESKD [1]. Despite improvements in understanding Cloxiquine the molecular mechanisms involving the development and progression of diabetic nephropathy, such as advanced glycation end-products, protein kinase C, and transforming growth factor- (TGF-) [2], certain effective therapeutic strategies remain to be established. Perhaps, multi-target therapy may be required for diabetic nephropathy treatment, and therefore, further identification of the potential therapeutic targets show great promise. Angiogenesis, the growth of new blood vessels from pre-existing vessels, is usually associated with a number of pathological processes, and is also involved in the pathogenesis of diabetic nephropathy. Previous studies exhibited new capillary formation and pre-existing capillary elongation [3, 4], as well as increased vascular endothelial growth factor (VEGF) level in diabetic glomeruli [5]. In addition, excessive activation of glomerular VEGF signaling in mice has been shown to cause mesangial matrix growth, resembling diabetic nephropathy [6, 7]. Since the landmark study that revealed the renoprotective efficacy of anti-VEGF antibody in diabetic mice [8], anti-angiogenic strategies remain possible options for diabetic nephropathy treatment. In contrast, concerns regarding anti-VEGF antibody-induced renal thrombotic microangiopathy have limited anti-VEGF strategies [9]. Considering the possibility that anti-angiogenic strategies could suppress glomerular lesions in diabetes, including increased capillary area and mesangial growth, novel angiogenic factors involved in the Cloxiquine pathogenesis of diabetic nephropathy are likely to become promising therapeutic targets. Vasohibin-1 (VASH1) is usually a unique endothelium-derived angiogenesis inhibitor, which prevents proliferation and migration of endothelial cells in an autocrine manner [10, 11]. We previously reported the therapeutic efficacy of adenoviral transfer of VASH1 in diabetic mice models [12, 13], and exacerbation of diabetic renal alterations in VASH1 heterozygous deficient mice [14], indicating the protective role of VASH1 in diabetic nephropathy. Vasohibin-2 (VASH2) was identified as a homolog to VASH1 [15]. In contrast to VASH1, VASH2 is known to possess pro-angiogenic activity [16]. Gene deletion of VASH2 Lum or neutralizing antibody against it has been shown to inhibit malignancy growth [17, 18]. Considering the above mentioned therapeutic effects of VASH1, VASH2 is usually expected to be a potential target for novel therapeutic strategy for diabetic nephropathy. Moreover, recent reports exhibited that VASH2 could enhance TGF- signaling in malignancy cells [19]. Therefore, reduced VASH2 expression can possibly lead to the prevention of TGF–mediated glomerular alterations. In the present study, we have exhibited the improvement of diabetic nephropathy in VASH2-deficient mice, and the inhibition of high glucose-induced extracellular matrix (ECM) protein production in cultured mesangial cells with suppressed VASH2 expression. Materials and methods Animals and experimental protocols VASH-2 homozygous knockout (VASH-2mice were fed a standard pellet laboratory chow and were provided with water (V2KO-NDM), (3) diabetic WT (WT-DM) and (4) diabetic VASH-2(V2KO-DM) mice. In Table 1 and Figs ?Figs11 and ?and2,2, we used the following; six for WT-NDM, six for V2KO-NDM, ten for WT-DM and eight for V2KO-DM mice. However, we used six mice for each group in the remaining experiments. Open in a separate windows Fig 1 Urine albumin excretion, renal hypertrophy and creatinine clearance in non-diabetic and diabetic wild-type and VASH2 knockout mice.(A) Six weeks after the induction of hyperglycemia, albuminuria in diabetic wild-type (WT) mice (solid circles) was significantly exacerbated compared with that in non-diabetic WT mice (open circles). Although no difference was found in albuminuria between non-diabetic WT and non-diabetic VASH2 knockout mice (open squares), increased albuminuria induced by hyperglycemia was markedly prevented in diabetic VASH2 knockout mice (solid squares). (B, C) The increase in kidney weight-to-body excess weight ratio (B) and urine volume (C).