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Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome.

Clement LC, Avila-Casado C, Macé C, Soria E, Bakker WW, Kersten S, Chugh SS - Nat. Med. (2010)

Bottom Line: In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease.Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria.When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%.

View Article: PubMed Central - PubMed

Affiliation: Glomerular Disease Therapeutics Laboratory, and Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4(-/-) mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.

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Characterization of male Angptl4 transgenic (TG) mice and rats. (a) Light microscopy (left panels) and confocal assessment of Angptl4 (middle panel) and ZO-1 (right panel) expression in Angptl4 transgenic and wild type (WT) mice. (b) Electron micrograph of 3 month old transgenic mouse glomeruli, showing intact (FP) and effaced podocyte foot processes (EFP). (c) Immunogold electron microscopy for Angptl4 showing gold particles in podocytes and glomerular basement membrane (GBM, arrows) in Angptl4 transgenic mice. (d) Proteinuria in 3 month old Angptl4 transgenic mice. (e) Rat Angptl4 transgenic constructs for the targeted expression of Angptl4 in podocytes (NPHS2-Angptl4, left panel) and adipose tissue (aP2-Angptl4, right panel) in rats. (f) Multi-organ mRNA expression profile of Angptl4 in podocyte specific and adipose tissue specific transgenic rats. (g) Periodic Acid Schiff stained sections from 3 month old wild type and heterozygous transgenic rats. Arrows point towards prominent podocytes in NPHS2-Angptl4 transgenic rats. (h) Confocal expression of Angptl4 (red) in NPHS2-Angptl4 transgenic rat glomeruli, and co-localization with podocyte protein nephrin (green, overlap yellow) and GBM heparan sulfate proteoglycan (blue, overlap fushia). (i) Electron micrograph of a glomerular capillary loop from a 5 month homozygous NPHS2-Angptl4 transgenic rat, showing diffuse foot process effacement (arrows). (j) Immunogold electron microscopy for Angptl4 in NPHS2-Angptl4 transgenic rats of increasing age (left to right), with transition from intact foot processes to foot process effacement (first noted around age 3 months), and clustering of gold particles in the GBM noted prominently in areas opposite to effaced foot processes (middle and right panels).Scale bars (a) 10 µm (b) 1 µm (c) 0.25 µm (g) 10 µm (h) 8µm (i) 1 µm (j) 0.2 µm. ENDO (endothelium). ** P < 0.01, *** P < 0.001
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Figure 2: Characterization of male Angptl4 transgenic (TG) mice and rats. (a) Light microscopy (left panels) and confocal assessment of Angptl4 (middle panel) and ZO-1 (right panel) expression in Angptl4 transgenic and wild type (WT) mice. (b) Electron micrograph of 3 month old transgenic mouse glomeruli, showing intact (FP) and effaced podocyte foot processes (EFP). (c) Immunogold electron microscopy for Angptl4 showing gold particles in podocytes and glomerular basement membrane (GBM, arrows) in Angptl4 transgenic mice. (d) Proteinuria in 3 month old Angptl4 transgenic mice. (e) Rat Angptl4 transgenic constructs for the targeted expression of Angptl4 in podocytes (NPHS2-Angptl4, left panel) and adipose tissue (aP2-Angptl4, right panel) in rats. (f) Multi-organ mRNA expression profile of Angptl4 in podocyte specific and adipose tissue specific transgenic rats. (g) Periodic Acid Schiff stained sections from 3 month old wild type and heterozygous transgenic rats. Arrows point towards prominent podocytes in NPHS2-Angptl4 transgenic rats. (h) Confocal expression of Angptl4 (red) in NPHS2-Angptl4 transgenic rat glomeruli, and co-localization with podocyte protein nephrin (green, overlap yellow) and GBM heparan sulfate proteoglycan (blue, overlap fushia). (i) Electron micrograph of a glomerular capillary loop from a 5 month homozygous NPHS2-Angptl4 transgenic rat, showing diffuse foot process effacement (arrows). (j) Immunogold electron microscopy for Angptl4 in NPHS2-Angptl4 transgenic rats of increasing age (left to right), with transition from intact foot processes to foot process effacement (first noted around age 3 months), and clustering of gold particles in the GBM noted prominently in areas opposite to effaced foot processes (middle and right panels).Scale bars (a) 10 µm (b) 1 µm (c) 0.25 µm (g) 10 µm (h) 8µm (i) 1 µm (j) 0.2 µm. ENDO (endothelium). ** P < 0.01, *** P < 0.001

Mentions: To study the biological role of Angptl4 upregulation in nephrotic syndrome, we first assessed Angptl4 expression in the podocyte in a previously published Angptl4 transgenic mouse model (13) (Fig. 2a–2d, Supplementary Fig. 2d,e). Glomeruli appeared normal on light microscopy (Fig. 2a). We noted increased glomerular expression of Angptl4, but not of another podocyte expressed protein Zona Occludens 1 (ZO-1) (Fig. 2a) in 3 month old transgenic mice. Angptl4 expression co-localized with podocyte expressed CD2AP (Supplementary Fig. 2d). Electron microscopy revealed 50% effacement or broadening of foot processes (Fig. 2b). immunogold electron microscopy revealed a large number of gold particles in podocyte foot processes, GBM and close to the endothelial cell surface (Fig. 2c), especially in areas opposite to foot process effacement (Fig. 2c, right panel). These mice had mild proteinuria (Fig. 2d).


Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome.

Clement LC, Avila-Casado C, Macé C, Soria E, Bakker WW, Kersten S, Chugh SS - Nat. Med. (2010)

Characterization of male Angptl4 transgenic (TG) mice and rats. (a) Light microscopy (left panels) and confocal assessment of Angptl4 (middle panel) and ZO-1 (right panel) expression in Angptl4 transgenic and wild type (WT) mice. (b) Electron micrograph of 3 month old transgenic mouse glomeruli, showing intact (FP) and effaced podocyte foot processes (EFP). (c) Immunogold electron microscopy for Angptl4 showing gold particles in podocytes and glomerular basement membrane (GBM, arrows) in Angptl4 transgenic mice. (d) Proteinuria in 3 month old Angptl4 transgenic mice. (e) Rat Angptl4 transgenic constructs for the targeted expression of Angptl4 in podocytes (NPHS2-Angptl4, left panel) and adipose tissue (aP2-Angptl4, right panel) in rats. (f) Multi-organ mRNA expression profile of Angptl4 in podocyte specific and adipose tissue specific transgenic rats. (g) Periodic Acid Schiff stained sections from 3 month old wild type and heterozygous transgenic rats. Arrows point towards prominent podocytes in NPHS2-Angptl4 transgenic rats. (h) Confocal expression of Angptl4 (red) in NPHS2-Angptl4 transgenic rat glomeruli, and co-localization with podocyte protein nephrin (green, overlap yellow) and GBM heparan sulfate proteoglycan (blue, overlap fushia). (i) Electron micrograph of a glomerular capillary loop from a 5 month homozygous NPHS2-Angptl4 transgenic rat, showing diffuse foot process effacement (arrows). (j) Immunogold electron microscopy for Angptl4 in NPHS2-Angptl4 transgenic rats of increasing age (left to right), with transition from intact foot processes to foot process effacement (first noted around age 3 months), and clustering of gold particles in the GBM noted prominently in areas opposite to effaced foot processes (middle and right panels).Scale bars (a) 10 µm (b) 1 µm (c) 0.25 µm (g) 10 µm (h) 8µm (i) 1 µm (j) 0.2 µm. ENDO (endothelium). ** P < 0.01, *** P < 0.001
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Related In: Results  -  Collection

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Figure 2: Characterization of male Angptl4 transgenic (TG) mice and rats. (a) Light microscopy (left panels) and confocal assessment of Angptl4 (middle panel) and ZO-1 (right panel) expression in Angptl4 transgenic and wild type (WT) mice. (b) Electron micrograph of 3 month old transgenic mouse glomeruli, showing intact (FP) and effaced podocyte foot processes (EFP). (c) Immunogold electron microscopy for Angptl4 showing gold particles in podocytes and glomerular basement membrane (GBM, arrows) in Angptl4 transgenic mice. (d) Proteinuria in 3 month old Angptl4 transgenic mice. (e) Rat Angptl4 transgenic constructs for the targeted expression of Angptl4 in podocytes (NPHS2-Angptl4, left panel) and adipose tissue (aP2-Angptl4, right panel) in rats. (f) Multi-organ mRNA expression profile of Angptl4 in podocyte specific and adipose tissue specific transgenic rats. (g) Periodic Acid Schiff stained sections from 3 month old wild type and heterozygous transgenic rats. Arrows point towards prominent podocytes in NPHS2-Angptl4 transgenic rats. (h) Confocal expression of Angptl4 (red) in NPHS2-Angptl4 transgenic rat glomeruli, and co-localization with podocyte protein nephrin (green, overlap yellow) and GBM heparan sulfate proteoglycan (blue, overlap fushia). (i) Electron micrograph of a glomerular capillary loop from a 5 month homozygous NPHS2-Angptl4 transgenic rat, showing diffuse foot process effacement (arrows). (j) Immunogold electron microscopy for Angptl4 in NPHS2-Angptl4 transgenic rats of increasing age (left to right), with transition from intact foot processes to foot process effacement (first noted around age 3 months), and clustering of gold particles in the GBM noted prominently in areas opposite to effaced foot processes (middle and right panels).Scale bars (a) 10 µm (b) 1 µm (c) 0.25 µm (g) 10 µm (h) 8µm (i) 1 µm (j) 0.2 µm. ENDO (endothelium). ** P < 0.01, *** P < 0.001
Mentions: To study the biological role of Angptl4 upregulation in nephrotic syndrome, we first assessed Angptl4 expression in the podocyte in a previously published Angptl4 transgenic mouse model (13) (Fig. 2a–2d, Supplementary Fig. 2d,e). Glomeruli appeared normal on light microscopy (Fig. 2a). We noted increased glomerular expression of Angptl4, but not of another podocyte expressed protein Zona Occludens 1 (ZO-1) (Fig. 2a) in 3 month old transgenic mice. Angptl4 expression co-localized with podocyte expressed CD2AP (Supplementary Fig. 2d). Electron microscopy revealed 50% effacement or broadening of foot processes (Fig. 2b). immunogold electron microscopy revealed a large number of gold particles in podocyte foot processes, GBM and close to the endothelial cell surface (Fig. 2c), especially in areas opposite to foot process effacement (Fig. 2c, right panel). These mice had mild proteinuria (Fig. 2d).

Bottom Line: In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease.Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria.When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%.

View Article: PubMed Central - PubMed

Affiliation: Glomerular Disease Therapeutics Laboratory, and Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4(-/-) mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.

Show MeSH
Related in: MedlinePlus