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

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

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

Enhanced accumulation of mesangial matrix and renal TGF-β/pSmad3 in the diabetic VASH1^+/− mice.

<p><i>A–D</i>: Representative light microscopic images of glomeruli (periodic acid-Schiff staining, original magnification x400) from 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. <i>E–H</i>: The glomerular accumulation of type IV collagen was assessed by the indirect immunofluorescence method for non-diabetic wild-type (<i>E</i>), non-diabetic VASH1^+/− (<i>F</i>), diabetic wild-type (<i>G</i>) and diabetic VASH1^+/− (<i>H</i>) mice. <i>E–H:</i> Original magnification x400. <i>I–K</i>: The increases in the glomerular volume, mesangial matrix index and type IV collagen induced by high glucose were exacerbated in the VASH1^+/− mice. The mesangial matrix index was defined as the proportion of the glomerular tuft occupied by the mesangial matrix area (excluding nuclei). The amount of immunoreactive type IV collagen in the glomeruli relative to the non-diabetic wild-type mice is shown (<i>K</i>). <i>L and M</i>: Immunoblots for TGF-β, phosphorylated Smad3 (pSmad3), Smad3 and actin are shown. <i>L (lower panel)</i>: The intensity of the TGF-β protein relative to actin is shown. <i>M (lower panel)</i>: The intensity of pSmad3 relative to Smad3 is 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>P</i><0.05 vs. non-diabetic wild-type or VASH1^+/− mice. ^†<i>P</i><0.05 vs. diabetic wild-type mice. The results of glomerular volume, type IV collagen score and immunoblots 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: DV, diabetic Vasohibin-1^+/− mice; DW, diabetic wild-type mice; NV, non-diabetic Vasohibin-1^+/− mice; NW, non-diabetic wild-type mice.</p

The influence of Vasohibin-1 knockdown on slit proteins and angiogenesis-related factors in cultured podocyte.

<p>Cells were cultured under normal glucose (NG; 5.5 mM), NG+Mannitol (normal D-glucose plus D-mannitol; 19.5 mM) or high glucose (HG; 25 mM) condition for 24 hours in the presence of control siRNA (siCon; 10 nM) or VASH1 siRNA (siV1; 10 nM). <i>A and B</i>: The amounts of Vasohibin-1 (VASH1) (<i>A</i>) and nephrin (<i>B</i>) mRNA relative to 18S rRNA are shown. <i>C–F</i>: Immunoblots for VASH1, ZO-1, VEGF-A, angiopoietin-1 (Ang-1) and actin are shown. In each lane, 20 µg of protein obtained from cultured mouse podocytes was loaded. The intensities of VASH1 (<i>C</i>), ZO-1 (<i>D</i>), VEGF-A (<i>E</i>) and Ang-1 (<i>F</i>) protein relative to actin are shown. ^§<i>P</i><0.05 vs. control siRNA (NG, NG+Mannitol (Manni) or HG). *<i>P</i><0.05 vs. control siRNA (NG or NG+Manni). ^‡<i>P</i><0.05 vs. control siRNA (HG). ^†P<0.05 vs. VASH1 siRNA (NG) or control siRNA (HG). ^#<i>P</i><0.05 vs. control siRNA (NG or NG+Manni) or VASH1 siRNA (NG). The results were expressed relative to the cells cultured with NG and control siRNA that were arbitrarily assigned a value of 100. Each column shows the mean ± SE. <i>n</i> = 4 for each group.</p

Correlations between the plasma and urinary levels of vasohibin-1-small vasohibin-binding protein complex and small vasohibin-binding protein, and the clinical/histological parameters and the plasma/urinary levels of vasohibin-1.

<p>Abbreviations: eGFR, estimated glomerular filtration rate; SVBP, small vasohibin-binding protein; U-Cr, urinary level of creatinine; VASH-1, vasohibin-1.</p>a<p><i>P</i><0.05.</p>b<p><i>P</i><0.01.</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

Accelerated podocyte injuries in the diabetic VASH1^+/− mice.

<p><i>A–D</i>: Immunofluorescent staining of nephrin. The distribution of nephrin 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–H</i>: Immunofluorescent staining of ZO-1. The distribution of ZO-1 was determined by an indirect immunofluorescence technique in non-diabetic wild-type (<i>E</i>), non-diabetic VASH1^+/− (<i>F</i>), diabetic wild-type (<i>G</i>) and diabetic VASH1^+/− (<i>H</i>) mice. Original magnification x400. <i>I–L</i>: TEM showed the ultrastructural features, including GBM thickening, foot process effacement and fusion in non-diabetic wild-type (<i>I</i>), non-diabetic VASH1^+/− (<i>J</i>), diabetic wild-type (<i>K</i>) and diabetic VASH1^+/− (L) mice. Asterisks, capillary lumen; arrows, foot process fusion. Scale bars, 1 µm. <i>M and N</i>: The staining scores for nephrin and ZO-1 are shown as “redistribution scores”. The staining patterns of nephrin and ZO-1 were evaluated using the method described in the MATERIALS AND METHODS. <i>O and P:</i> The TEM morphometry of the GBM thickness and slit-diaphragm density. *<i>P</i><0.05 vs. non-diabetic wild-type or non-diabetic VASH1^+/− mice. ^†<i>P</i><0.05 vs. diabetic wild-type mice. Each column shows the mean ± SE. <i>n</i> = 4 for each group. Abbreviations: GBM, glomerular basement membrane; TEM, transmission electron microscopy; VASH1^+/−, Vasohbin-1^+/− mice; Wild, wild-type mice.</p

The results of the Kaplan-Meier analysis of the composite renal endpoint.

<p>A composite renal event was defined as a decline in the eGFR of more than 30% of the baseline value, initiation of renal replacement therapy or death associated with a renal disorder. In order to perform a Kaplan-Meier analysis of the plasma and urinary levels of VASH-1, the patients were stratified into two groups using the median (609 fmol/mL for the plasma level and 21 fmol/mg for the urinary level of VASH-1) as the cutoff point. The log-rank test was used to compare differences between the two groups. (A and B) Increased plasma levels of VASH-1 were significantly correlated and the urinary levels of VASH-1 tended to be correlated with worse renal outcomes. (C) In order to perform a Kaplan-Meier analysis of the plasma levels of the VASH-1-SVBP complex, the patients were stratified into three groups (<316, 316 to 408 and >408 fmol/mL). The group with the highest plasma levels of the VASH-1-SVBP complex exhibited significantly worse renal outcomes than the groups with moderate or low plasma levels. Abbreviations: eGFR, estimated glomerular filtration rate; RRT, renal replacement therapy, SVBP, small vasohibin-binding protein; VASH-1, vasohibin-1.</p

Results of the multivariate logistic analysis of the risk of composite renal events.

<p>Abbreviations: CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio; SVBP, small vasohibin-binding protein; U-Cr, urinary level of creatinine; VASH-1, vasohibin-1. A composite renal event was defined as a decline in the eGFR of more than 30% of the baseline value, initiation of renal replacement therapy or death associated with a renal disorder. In order to perform a multivariate logistic analysis of the plasma and urinary levels of VASH-1, the patients were stratified into two groups using the median value as the cutoff point, and the plasma levels of the VASH-1-SVBP complex, the patients were stratified into three groups.</p><p>Model 1: adjusted for age and gender.</p><p>Model 2: adjusted for age, gender and systolic blood pressure.</p