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

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.

Figure 1

Angptl4 mRNA and protein expression in experimental glomerular disease. (a) Induction of proteinuria in rats 24 hours after injection of γ2-NTS. (b) Upregulation of glomerular Angptl4 mRNA expression in rats injected with γ2-NTS. (c) Proteinuria in Angptl4^−/− and Angptl4^+/+ mice after injection of lipopolysaccharide (LPS). (d) Confocal expression of Angptl4 protein in rat glomeruli, and colocalization with podocyte protein CD2AP. Absorbing out reactivity from anti-Angptl4 antibody with recombinant Angptl4 abolished immunoreactivity. (e) Assessment of changes in glomerular Angptl4 expression in rat models of minimal change disease (puromycin nephrosis, PAN), membranous nephropathy (passive Heymann nephritis, PHN), mesangial injury (anti-Thy1.1 nephritis), and severe focal and segmental glomerulosclerosis (non-HIV collapsing glomerulopathy, CG). Threshold for significance was 3-fold change. (f) Confocal assessment of Angptl4 expression (red) in control and PAN Day 6 glomeruli, and co-localization with GBM heparan sulfate proteoglycan (white, top and middle panels) and podocyte protein nephrin (green, overlap yellow, bottom panel). (g) Immunogold electron microscopy of PAN Day 6 rat glomeruli to demonstrate Angptl4 expression (gold particles) in the podocytes (yellow arrows) and GBM (black arrows). Scale bars (d) 7.5 µm (f) 10 µ(g) 0.33 µm. NTS (nephrotoxic serum), CD2AP (CD2 adapter protein), LCM (laser capture microdissection), GBM (glomerular basement membrane), EFP (effaced foot processes), Endo (endothelium).

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

Figure 3

Relationship of Angptl4 overexpression with proteinuria. (a) Albuminuria in female NPHS2-Angptl4 transgenic (TG) rats. (b) Albuminuria in male heterozygous NPHS2-Angptl4 transgenic rats (c) Albuminuria in male homozygous NPHS2-Angptl4 transgenic rats. (d) GelCode blue stained SDS PAGE of urinary protein from transgenic rats, rats with PAN, and individuals with minimal change disease (MCD) and membranous nephropathy (MN). Arrow points towards prominent 70 kDa intact albumin band. Mean percentage densitometry of intact albumin is shown for each lane (details in Supplementary Fig. 3d). (e) Proteinuria following induction of low dose PAN in heterozygous male NPHS2-Angptl4 transgenic and wild type littermates. (f) Proteinuria in Wistar rats treated with glucocorticoids (PAN-S) or PBS (PAN) on alternate days starting 1 day after induction of PAN. (g) Glomerular Angptl4 mRNA expression in PAN rats described in panel f. * P<0.05; ** P < 0.01, *** P<0.001.

Figure 4

Relationship of Angptl4 sialylation with proteinuria. (a) Two-dimensional (2D) gel electrophoresis and Western blot of protein from perfused glomeruli show neutral and high pI low order Angptl4 oligomers (pink, orange arrows) in control, PAN Day 6, and glucocorticoid treated PAN Day 6 rats (from experiment in Fig. 3f). Reactivity of sialic acid binding lectin MAA to these oligomers was also assessed (exemplified for PAN, excerpts from independent blots). (b) Densitometry of total, neutral and high pI oligomers shown in panel a. (c) 2D gel electrophoresis and Western blot of concentrated supernatant from Angptl4 – HEK293 stable cell line incubated with ManNAc or control, and analyzed for Angptl4 expression and binding with sialic acid binding lectin MAA. Green arrow and line highlight high pI protein in the control treated group, whereas blue arrow in the ManNAc treated group shows neutral pI protein. (d) Same study as panel c, except done with supernatant from the GEC stable cell line. (e) 2D gel electrophoresis and Western blot studies of glomerular protein from NPHS2-Angptl4 transgenic rats given tap water or tap water with ManNAc for 12 days. Blots were analyzed for neutral pI (enclosed in red ovals) and high pI (enclosed in green ovals) Angptl4 using anti-Angptl4 antibody and sialic acid binding lectin Sambucus Nigra (SNA I). (f) Percentage of neutral and high pI Angptl4 within each group as assessed by densitometry. (g) Albuminuria in NPHS2-Angptl4 transgenic rats given tap water (Control group) or tap water with ManNAC (Treatment group) for 12 days (Treatment group, ManNAC phase), followed by plain tap water for 24 days (Treatment group, Washout phase). Values are expressed as a percentage of the baseline albuminuria (designated as 100%). Individual tracings are shown in Supplementary Fig. 8. In panel b, all * differences are with control values. In panel g, all * differences are with baseline values. Loading controls shown in Supplementary Fig. 6. * P<0.05; ** P<0.01; *** P<0.001.