Localization of endogenous VASH2 in glomeruli.

<p>(A) The mRNA level of VASH2 in the kidney cortex from wild-type mice was assessed by real-time PCR. VASH2 expression was increased in diabetic condition compared with non-diabetic mice kidney. n = 6 for each group. *<i>P</i><0.05 versus wild-type mice. (B) VASH2 expression is detected with immunofluorescence for β-galactosidase. No immunoreactivity in glomeruli is seen in wild-type mice (left panel), whereas glomeruli from non-diabetic and diabetic VAHS2 knockout (VASH2^{<i>LacZ/LacZ</i>}) mice (middle and right panel, respectively) show a β-galactosidase-positive area (original magnification, ×400). (C) Double immunofluorescence for β-galactosidase and markers for glomerular component cells in VASH2 knockout mice show that the localization of β-galactosidase-positive area are consistent with platelet-derived growth factor receptor-β (PDGFRβ)-positive mesangial cells, but not CD31-positive endothelial cells and zonula occludens-1 (ZO-1)-positive podocytes.</p

Characteristics of non-diabetic and diabetic wild-type and VASH2-deficient mice at the end of study.

<p>Characteristics of non-diabetic and diabetic wild-type and VASH2-deficient mice at the end of study.</p

Alterations of glomerular endothelial area and VEGF-A expression in diabetic VASH2 knockout mice.

<p>(A) The distribution of CD31, a marker for endothelial cells, was determined by immunofluorescence in non-diabetic wild-type, non-diabetic VASH2 knockout, diabetic wild-type, and diabetic VASH2 knockout mice (original magnification, ×400). (B) In quantitative analysis, CD31-positive glomerular endothelial area was expanded in diabetic wild-type mice, but it was significantly prevented in diabetic VASH2 knockout mice. No difference was found in endothelial area between non-diabetic wild-type and non-diabetic VASH2 knockout mice. (C, D) Immunoblot for vascular endothelial growth factor-A (VEGF-A; C) and VEGF receptor-2 (VEGFR2; D). Each lane was loaded with 40 μg of protein obtained from the renal cortex. Each band was scanned and subjected to a densitometric analysis. Increased VEGF-A level induced by diabetes showed no difference between wild-type and VASH2 knockout mice, whereas increased VEGFR2 expression seen in diabetic wild-type mice was significantly suppressed in diabetic VASH2 knockout mice. n = 6 for each group. *<i>P</i><0.05 versus non-diabetic WT or VASH2 knockout mice, ^#<i>P</i><0.05 versus diabetic WT mice. Each column shows the mean ± SE.</p

Urine albumin excretion, renal hypertrophy and creatinine clearance in non-diabetic and diabetic wild-type and VASH2 knockout mice.

<p>(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 weight ratio (B) and urine volume (C) induced by hyperglycemia did not significantly differ between WT and VASH2 knockout mice. (D) The increase in creatinine clearance (Ccr) level induced by hyperglycemia was significantly prevented in VASH2 knockout mice compared with WT mice. n = 6 for non-diabetic WT, 6 for non-diabetic VASH2 knockout, 10 for diabetic WT, and 8 for diabetic VASH2 knockout mice. *<i>P</i><0.05 versus non-diabetic WT or VASH2 knockout mice, **<i>P</i><0.01 versus non-diabetic WT or VASH2 knockout mice, ^#<i>P</i><0.05 versus diabetic WT mice, ^##<i>P</i><0.01 versus diabetic WT mice. Each column shows the mean ± SE.</p

Histological and ultrastructural alterations in diabetic VASH2 knockout mice.

<p>(A) Representative light microscopic images of glomeruli from non-diabetic wild-type, non-diabetic VASH2 knockout, diabetic wild-type, and diabetic VASH2 knockout mice (periodic acid-Schiff staining, original magnification, ×400). (B, C) Diabetes-induced increases in glomerular volume (B) and mesangial matrix index (C) were significantly prevented in VASH2 knockout mice compared with wild-type mice. The mesangial matrix index was defined as the proportion of the glomerular volume occupied by mesangial matrix area (excluding nuclei). (D) Representative transmission electron microscopic images of glomerular capillary tufts from non-diabetic wild-type, non-diabetic VASH2 knockout, diabetic wild-type, and diabetic VASH2 knockout mice (scale bars, 2 μm). Diabetes caused foot process fusion (arrow) and obscured silt-diaphragms. (E) Increased GBM thickness in diabetic wild-type mice was significantly suppressed in diabetic VASH2 knockout mice. (F) The decrease in slit diaphragm density observed in diabetic wild-type mice was also improved in diabetic VASH2 knockout mice. n = 6 for non-diabetic WT, 6 for non-diabetic VASH2 knockout, 10 for diabetic WT, and 8 for diabetic VASH2 knockout mice. *<i>P</i><0.05 versus non-diabetic WT or VASH2 knockout mice, ^#<i>P</i><0.05 versus diabetic WT mice. Each column shows the mean ± SE.</p

The role of VASH2 knockdown in extracellular matrix production in cultured mesangial cells.

<p>Human mesangial cells (HMCs) were cultured under normal glucose (NG, 5.5 mM), NG plus mannitol (MN; NG plus mannitol, 19.5 mM) or high glucose (HG, 25 mM) condition for 24 hours in the presence of negative control siRNA (siCont, 10 nM) or VASH2 siRNA (siVASH2, 10 nM). (A) The expression of VASH2 mRNA relative to 18S rRNA was elevated in HG but not in MN condition. (B, C) Immunoblots for VASH2 and β-actin are shown (B). Each lane was loaded with 20 μg of protein obtained from cultured HMCs. Each band was scanned and subjected to a densitometric analysis (C). Transfection of siVASH2 reduced the level of VASH2 by approximately 80%. (D, E) The amount of type IV collagen α3 (D) and connective tissue growth factor (CTGF; E) relative to 18S rRNA is shown. Increase in these mRNA level caused by HG condition was significantly prevented by transfection of siVASH2. n = 4 for each group. ^§<i>P</i><0.05 versus NG or MN, *<i>P</i><0.05 versus NG with siCont or NG with siVASH2, ^#<i>P</i><0.05 versus HG with siCont. Each column shows the mean ± SE.</p

The alterations of endothelial cells and angiogenic factors in the diabetic VASH1^+/− mice.

<p><i>A–D</i>: The distribution of CD31, a marker for endothelial cells, was determined by an indirect immunofluorescence technique in non-diabetic wild-type (<i>A</i>), non-diabetic VASH1^+/− (<i>B</i>), diabetic wild-type (<i>C</i>) and diabetic VASH1^+/− (<i>D</i>) mice. Original magnification x400. <i>E</i>: The glomerular CD31⁺ endothelial area was quantitated. <i>F</i>: The CD31⁺ peritubular capillary density was quantitated. <i>G–I</i>: Immunoblots for VEGF-A, angiopoietin (Ang)-1, Ang-2 and actin are shown. Each lane was loaded with 50 µg of protein obtained from the renal cortex. Each band was scanned and subjected to a densitometric analysis. <i>G (lower panels)</i>: The intensity of the VEGF-A protein relative to actin is shown. <i>H (lower panels)</i>: The intensity of Ang-1 relative to actin is shown. <i>I (lower panels)</i>: The intensity of Ang-2 relative to actin is shown. *<i>P</i><0.05 vs. non-diabetic wild-type or VASH1^+/− mice. ^†<i>P</i><0.05 vs. diabetic wild-type mice. ^#<i>P</i><0.05 vs. non-diabetic wild-type mice. ^§<i>P</i><0.05 vs. non-diabetic VASH1^+/− or diabetic wild-type mice. ^‡<i>P</i><0.05 vs. non-diabetic wild-type, non-diabetic VASH1^+/− or diabetic wild-type mice. The results are expressed relative to non-diabetic wild-type mice that were arbitrarily assigned a value of 100. Each column shows the mean ± SE. <i>n</i> = 4 for each group. Abbreviations: VASH1^+/−, Vasohibin-1^+/− mice; Wild, wild-type mice.</p

Urinary and Plasma Levels of Vasohibin-1 Can Predict Renal Functional Deterioration in Patients with Renal Disorders

<div><p>Vasohibin-1 (VASH-1) is a negative feedback regulator of angiogenesis, and a small vasohibin-binding protein (SVBP) serves as its secretory chaperone and contributes to its antiangiogenic effects. In the present study, we aimed to define the clinical significance of VASH-1 and SVBP in patients with chronic kidney disease (CKD). We recruited 67 Japanese hospitalized patients with renal disorders with (n = 45) or without (n = 22) renal biopsy samples and 10 Japanese healthy controls. We evaluated the correlations between the plasma and urinary levels of VASH-1/VASH-1-SVBP complex/SVBP and the clinicopathological parameters. The plasma levels of VASH-1 were inversely correlated with age and systolic and diastolic blood pressure and positively correlated with crescent formation. Increased plasma and urinary levels of VASH-1 and VASH-1-SVBP complex were significantly correlated with worse renal outcomes. These results demonstrate an association between elevated urinary and plasma levels of VASH-1 and progressive decline of the renal function, thus suggesting a potential role for VASH-1 in predicting a worse renal prognosis in patients with renal disease, including CKD.</p></div

The baseline characteristics of the patients with or without renal biopsy, classified by the plasma levels of vasohibin-1.

<p>Abbreviations: eGFR, estimated glomerular filtration rate; RB, renal biopsy; SBP, systolic blood pressure; sCr, serum creatinine; VASH-1, vasohibin-1. The values are expressed as the means ± SD.</p>a<p><i>P</i><0.05 versus the Low group.</p>b<p><i>P</i><0.01 versus the Low group.</p

Correlations between the plasma and urinary levels of vasohibin-1, the vasohibin-1-small vasohibin-binding protein complex and small vasohibin-binding protein, and the annual change rates in the estimated glomerular filtration rate.

<p>Abbreviations: eGFR, estimated glomerular filtration rate; SVBP, small vasohibin-binding protein; U-Cr, urinary level of creatinine; VASH-1, vasohibin-1. The annual change rates in eGFR were evaluated as follows: the annual change in eGFR was divided by the eGFR at baseline.</p>a<p><i>P</i><0.05.</p