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

Nature Medicine volume 17, pages 117–122 (2011)Cite this article

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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 responses1. 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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Figure 1: Angptl4 mRNA and protein expression in experimental glomerular disease.
Figure 2: Characterization of male Angptl4 transgenic mice and rats.
Figure 3: Relationship between Angptl4 overexpression and proteinuria.
Figure 4: Relationship between Angptl4 sialylation and proteinuria.

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Acknowledgements

We thank B. Spiegelman (Dana-Farber Cancer Institute) for the mouse aP2 promoter construct; L. Holzman (University of Michigan) for the human NPHS2 promoter construct; P. Mundel (University of Miami) for mouse GECs; T. van Kuppevelt (Radboud University Nijmegen Medical Center) for the heparan sulfate proteoglycan–specific antibody p1113; D. Salant (Boston University) for γ2-NTS; and A. Köster (Eli Lilly) for Angptl4−/− mice. We also thank G. Bai for the generation of probe for in situ hybridization; S. Shastry for some ELISA assays for rat and mouse albumin; S. Rahmanuddin for selected immunogold studies and polyethyleneimine studies; F. Mattijssen for mouse LPS study; J. Novak for advice on choice of lectins; and Y.S. Kanwar and M. Venkatachalam for useful discussions on heparan sulfate proteoglycans. Supported by the US National Institutes of Health (R56DK077073, R01DK077073 to S.S.C.), Norman S. Coplon Satellite Research Grant to S.S.C. and CONACYT 111 grant to C.A.-C. We thank the University of Alabama at Birmingham–University of California–San Diego George O'Brien Center Core C for measuring mouse urine creatinine by mass spectrometry. The use of sialic acid precursors, including ManNAc, to treat proteinuria and nephrotic syndrome is covered by US Provisional Application 61/351,865 filed by S.S.C.

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Author notes

  1. Carmen Avila-Casado and Camille Macé: These authors contributed equally to this work.

Authors and Affiliations

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

    Lionel C Clement, Camille Macé & Sumant S Chugh

  2. Department of Pathology, Instituto Nacional de Cardiologia, Mexico City, Mexico

    Carmen Avila-Casado & Elizabeth Soria

  3. Department of Pathology and Medical Biology, University Medical Center of Groningen, Groningen, The Netherlands

    Winston W Bakker

  4. Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands

    Sander Kersten

Authors
  1. Lionel C Clement
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Contributions

L.C.C. was lead postdoctoral fellow, conducted most of the animal studies, generated stable cell lines and purified recombinant protein, maintained transgenic rats and conducted all studies on transgenic rats, confocal imaging and in situ hybridization, most gene expression studies and selected two-dimensional gel studies. C.A.-C. interpreted and analyzed light microscopy, electron microscopy and immunogold electron microscopy sections for the study and conducted studies on induction of collapsing glomerulopathy in rats. C.M. conducted most of the two-dimensional gel electrophoresis and proteomic work, and most of the albumin ELISA assays. E.S. acted as electron microscopist and morphometrics expert, prepared tissue for electron microscopy, conducted and imaged conventional and most immunogold electron microscopy studies, and conducted alcian blue charge studies. W.W.B. provided human sera and assisted in study design and preparation of the manuscript. S.K. conducted studies on Angptl4 transgenic mice, provided tissue for histological and gene expression analysis, conducted Angptl4−/− mouse studies with NTS and made substantial contributions to the preparation of the manuscript. S.S.C. acted as senior investigator, planned and supervised the study, generated constructs for transgenic rats, conducted molecular biology and gene expression studies, conducted early animal studies, and wrote and revised the manuscript with input from the other authors.

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Correspondence to Sumant S Chugh.

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Supplementary Figures 1–11, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 1867 kb)

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Clement, L., Avila-Casado, C., Macé, C. et al. Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome. Nat Med 17, 117–122 (2011). https://doi.org/10.1038/nm.2261

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