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

Suppression of Adiponectin by Aberrantly Glycosylated IgA1 in Glomerular Mesangial Cells In Vitro and In Vivo

<div><p>The pathogenesis of IgA nephropathy (IgAN) may be associated with the mesangial deposition of aberrantly glycosylated IgA1. To identify mediators affected by aberrantly glycosylated IgA1 in cultured human mesangial cells (HMCs), we generated enzymatically modified desialylated and degalactosylated (deSial/deGal) IgA1. The state of deglycosylated IgA1 was confirmed by lectin binding to <em>Helix aspersa</em> (HAA) and <em>Sambucus nigra</em> (SNA). In the cytokine array analysis, 52 proteins were upregulated and 34 were downregulated in HMCs after stimulation with deSial/deGal IgA1. Among them, the secretion of adiponectin was suppressed in HMCs after stimulation with deSial/deGal IgA1. HMCs expressed mRNAs for adiponectin and its type 1 receptor, but not the type 2 receptor. Moreover, we revealed a downregulation of adiponectin expression in the glomeruli of renal biopsy specimens from patients with IgAN compared to those with lupus nephritis. We also demonstrated that aberrantly glycosylated IgA1 was deposited in the mesangium of patients with IgAN by dual staining of HAA and IgA. Moreover, the urinary HAA/SNA ratio of lectin binding was significantly higher in IgAN compared to other kidney diseases. Since adiponectin has anti-inflammatory effects, including the inhibition of adhesion molecules and cytokines, these data suggest that the local suppression of this adipokine by aberrantly glycosylated IgA1 could be involved in the regulation of glomerular inflammation and sclerosis in IgAN.</p> </div

Cytokine array analysis after stimulation of cultured human mesangial cells (HMCs) with native IgA or deSial/deGal IgA1.

<p>The HMCs were stimulated with native (A) or deSial/deGal IgA1 (50 µg/ml) (B) for 48 h. The culture supernatants were then applied for a protein array analysis. After incubation of samples with array membranes for 2 h at room temperature, the spots on the membranes were scanned and digitized. The signal intensities of the spots obtained from two separate experiments were analyzed. (The proteins that were up- or downregulated by approximately 2 fold are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033965#pone-0033965-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033965#pone-0033965-t002" target="_blank">2</a>.) The spots shown by arrows correspond to adiponectin. The intensity of the spots in the membrane stimulated with native IgA was higher than that of cells stimulated with deSial/deGal IgA1 in HMC (A, B).</p

The expression of adiponectin, AdipoR1 and AdipoR2 genes in cultured human mesangial cells (HMCs).

<p>(A) The cDNA from human adipocytes was utilized as a positive control for adiponectin and the cDNA from human hepatocytes was used as a control for AdipoR1 and AdipoR2. RT-PCR for adiponectin in HMCs after stimulation with either native or deSial/deGal IgA1 afforded cDNA bands of the same size (256 bp) as that amplified from human adipocyte cDNA. RT-PCR for AdipoR1 in HMCs afforded cDNA bands of the same size (70 bp) as that amplified from human hepatocytes. The HMCs did not express AdipoR2 mRNA. The expression of the GAPDH gene was used as an internal standard (350 bp). (B) The densitometric analysis of the expression of each cDNA.</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

The ELISA of total (A, C, D) and HMW (B, E) adiponectin after stimulation of HMCs with native or deSial/deGal IgA1.

<p>In HMCs, native IgA upregulated the total (A) and high molecular weight (HMW) (B) adiponectin release to the supernatant in a dose- (A, B) and time-dependent manner (C). Native IgA also increased the total and HMW adiponectin concentration in cell lysates in a dose-dependent manner (D, E). HMCs were cultured with different concentrations of native or deSial/deGal IgA1 (0, 6, 12.5, 25 or 50 µg/ml) for 48 h. For the time course study, HMCs were cultured with 25 µg/ml of native or deSial/deGal IgA1 for 0, 1, 6, 24 or 48 h. The concentrations of adiponectin were expressed as the means ± SE. Open diamonds, native IgA; closed circles, deSial/deGal IgA1. *<i>P</i> = 0.05 vs. medium control; #<i>P</i> = 0.01, native IgA vs. deSial/deGal IgA1.</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

The urinary HAA/SNA binding ratio in patients with IgAN and other kidney diseases.

<p>The HAA/SNA ratios were determined by the HAA or SNA lectin binding assays using anti-IgA antibody-coated plates. The HAA/SNA ratio was higher in IgAN patients compared to patients with other kidney diseases (OKD) (<i>P</i><0.05) (A). The level of HAA binding corrected for the urinary creatinine concentration was also higher in IgAN patients compared to patients with OKD (<i>P</i><0.05) (B). Each column consists of the means ± SE. OKD, <i>n</i> = 142; IgAN, <i>n</i> = 78 *<i>P</i><0.05.</p

The expression of adiponectin, αSMA and vWF in human renal biopsies.

<p>Immunofluorescent staining of renal biopsy specimens from patients with minor glomerular abnormalities (MGA; A, D, G, J, M and P), lupus nephritis (LN; B, E, H, K, N and Q) and IgA nephropathy (IgAN; C, F, I, L, O and R) are shown. (Upper panels) Each section was stained for αSMA (a marker of activated mesangial cells, red) (A–C) and adiponectin (green) (D–F). Some αSMA-positive cells were colocalized with adiponectin-positive cells (yellow) (G–I). Double positive areas were predominant in the glomeruli of LN patients (H, arrows) as compared to IgAN patients (I, arrows). (Lower panels) Each section was stained for vWF (a marker of endothelial cells, red) (J–L) and adiponectin (green) (M–O). Some vWF-positive cells were colocalized with adiponectin-positive cells (yellow) (P–R, arrowheads). Double positive areas predominated in the glomeruli of MGA patients (P). Strong and segmental staining of adiponectin was recognized in the glomeruli of LN patients (E and N). The adiponectin staining in the glomeruli of IgAN patients was weaker than that of LN and MGA patients. Scale bars represent 100 µm.</p

The profiles of the patients included in the analysis of urine samples.

<p>OKD, other kidney disease; IgAN, IgA nephropathy; eGFR, estimated glomerular filtration rate; RBC, red blood cell; HAA, <i>Helix aspersa</i>; SNA, <i>Sambucus nigra agglutinin</i>.</p>a<p>: <i>P</i><0.01,</p>b<p>: <i>P</i><0.05.</p