Heymann Nephritis in Lewis Rats

Yuan Min Wang1, Vincent W.S. Lee2, Huiling Wu3, David C.H. Harris2, Stephen I. Alexander1

1 Centre for Kidney Research, Children's Hospital at Westmead, Sydney, New South Wales, 2 Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Sydney, New South Wales, 3 Collaborative Transplant Research Group, Royal Prince Alfred Hospital, Sydney, New South Wales
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.29
DOI:  10.1002/0471142735.im1529s109
Online Posting Date:  April, 2015
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Human membranous nephritis is a major cause of end‐stage kidney disease. Active Heymann nephritis (HN) is an auto‐immune model of membranous nephritis induced in Lewis rats by immunization with a crude renal tubular antigen (Fx1A) or megalin (gp330). The pathogenesis of HN is through the binding of anti‐Fx1A autoantibodies to the auto‐antigen expressed on glomerular epithelial cells, resulting in severe glomerular injury and proteinuria. The pathological features of HN include immune deposits in glomeruli and infiltration of glomeruli and the tubulointerstitium by macrophages and T cells. This unit describes the method of the preparation of Fx1A and the induction of HN in Lewis rats by immunization with Fx1A. © 2015 by John Wiley & Sons, Inc.

Keywords: active Heymann nephritis; Lewis rats; Fx1A; proteinuria; glomerulonephritis

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 10 Sprague‐Dawley (SD) rats (male; 8 weeks old; 200 to 220 g)
  • Saline or PBS, pH 7.3 (e.g., Life Technologies), 25°C
  • Lewis rats (male; 8 weeks old; 180 to 200 g)
  • Mycobacterium tuberculosis HRa37 (Difco)
  • Incomplete Freund's adjuvant (IFA) (Sigma‐Aldrich)
  • Complete Freund's adjuvant (CFA) (Sigma‐Aldrich)
  • 1% (w/v) deoxycholic acid
  • Ammonium sulfate
  • 0.9% (w/v) NaCl
  • 10% (v/v) neutral‐buffered formalin (Sigma)
  • Periodic acid‐Schiff's (PAS) reagent and hematoxylin (Sigma‐Aldrich)
  • Paraffin
  • Glutaraldehyde solution (Karnovsky medium; EMS, cat. no. 15720; optional)
  • 150 mesh steel sieve, bore size 104‐106 μm (e.g., Interpath)
  • Bachman ultracentrifuge and TFT70 rotor, serial number 14790
  • Lyophilizer
  • 2‐ml glass syringes with luer locks for preparing the Fx1A emulsion (Pacific Lab Products; cat. no. 210/2)
  • ELISA "Ensemble" kit for the detection of rat primary antibody (e.g., Alpha Diagnostic International) or equivalent (see unit 2.1)
  • Hypodermic needles
  • Microplate spectrophotometer (e.g., Multiskan Ascent, Pathtech)
  • Metabolic cages
  • Automated chemistry analyzer (e.g. VITROS, Ortho Clinical Diagnostics, Johnson & Johnson)
  • Delta Vision Core Microscope (Applied Precision)
  • C96 Maxisorp MicroWell plates (e.g., NUNC, Thermo Fisher Scientific)
  • Additional reagents and equipment for euthanasia (unit 1.8), parenteral injections (unit 1.6), ELISA (unit 1.6), and blood collection by cardiac puncture (unit 1.7)
NOTE: Maintain all rats in a dedicated animal research facility, free of pathogens.
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Literature Cited

Literature Cited
  Cheng, I.K., Dorsch, S.E., and Hall, B.M. 1984. Lymphocyte subsets in Heymann nephritis. Lab. Invest. 51:286‐291.
  Farquhar, M.G., Saito, A., Kerjaschki, D., and Orlando, R.A. 1995. The Heymann nephritis antigenic complex: megalin (gp330) and RAP. J. Am. Soc. Nephrol. 6:35‐47.
  Heymann, W., Hackel, D.B., Harwood, S., Wilson, S.G., and Hunter, J.L. 1959. Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions. Proc. Soc. Exp. Biol. Med. 100:660‐664.
  Mizuno, S., Kurosawa, T., Matsumoto, K., Mizuno‐Horikawa, Y., Okamoto, M., and Nakamura, T. 1998. Hepatocyte growth factor prevents renal fibrosis and dysfunction in a mouse model of chronic renal disease. J. Clin. Invest. 101:1827‐1834.
  Quiza, C.G., Leenaerts, P.L., and Hall, B.M. 1992. The role of T cells in the mediation of glomerular injury in Heymann's nephritis in the rat. Int. Immunol. 4:423‐432.
  Walters, G., Wu, H., and Knight, J.F. 2001. Glomerular T cells in Heymann nephritis. Clin.Exp.Immunol. 126:319‐325.
  Wang, Y.M., Zhang, G.Y., Hu, M., Polhill, T., Sawyer, A., Zhou, J.J., Saito, M., Watson, D., Wu, H., Wang, Y., Wang, X.M., Harris, D.C., and Alexander, S.I. 2012. CD8+ Regulatory T Cells Induced by T Cell Vaccination Protect Against Autoimmune Nephritis. J. Am. Soc. Nephrol. 23(6):1058‐1067.
  Wu, H., Walters, G., Knight, J.F., and Alexander, S.I. 2003. DNA vaccination against specific pathogenic TCRs reduces proteinuria in active Heymann nephritis by inducing specific autoantibodies. J. Immunol. 171:4824‐4829.
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