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 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

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

Correlations between the plasma/urinary levels of vasohibin-1 and the clinical parameters.

<p>(A) Correlations between the plasma/urinary levels of VASH-1 and age. (B) Correlation between the urinary VASH-1/Cr ratio and age. (C) Correlation between the plasma level of VASH-1 and systolic blood pressure. (D) Correlation between the urinary VASH-1/Cr ratio and systolic blood pressure. (E) Correlation between the plasma level of VASH-1 and diastolic blood pressure. (F) Correlation between the urinary VASH-1/Cr ratio and diastolic blood pressure. (G) Correlation between the plasma level of VASH-1 and eGFR. (H) Correlation between the urinary VASH-1/Cr ratio and eGFR. (I) Correlation between the plasma level of VASH-1 and proteinuria. (J) Correlation between the urinary VASH-1/Cr ratio and proteinuria. Abbreviations: BP, blood pressure; eGFR, estimated glomerular filtration rate; U-Cr, urinary level of creatinine; VASH-1, vasohibin-1.</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