Adriamycin Nephropathy in BALB/c Mice

Yuan Min Wang1, Yiping Wang2, David C. H. Harris2, Stephen I. Alexander1, Vincent W. S. Lee2

1 Centre for Kidney Research, Children's Hospital at Westmead, Sydney, 2 Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Sydney
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.28
DOI:  10.1002/0471142735.im1528s108
Online Posting Date:  February, 2015
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Chronic proteinuric renal injury is a major cause of end stage renal disease. Adriamycin nephropathy (AN) is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease. This unit describes the method of AN induced by a single injection of adriamycin (ADR) in BALB/c mice. After the initial toxic injury, an immune‐mediated chronic proteinuric renal disease that resembles human focal segmental glomerulosclerosis develops. The clinic pathological features of AN are nephrotic syndrome, focal glomerulosclerosis, tubular injury, and interstitial compartment expansion with mononuclear cell infiltrates that are composed largely of macrophages and T cells. © 2015 by John Wiley & Sons, Inc.

Keywords: adriamycin (ADR); adriamycin nephropathy (AN); BALB/c mice; proteinuria; glomerulosclerosis

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Male BALB/c mice, 8 weeks old (20 g)
  • Adriamycin (ADR; doxorubicin hydrochloride; 10 mg/5 ml per vial as a stock solution, Pfizer Australia)
  • 10% neutral‐buffered formalin (Sigma‐Aldrich)
  • Colorimetric assay kit (Bio‐Rad, Hercules)
  • Paraffin
  • Periodic acid‐Schiff (PAS) kit (Sigma‐Aldrich)
  • Optimal cutting temperature (OCT) compound (Sakura Fintek)
  • Karnovsky's fixative (see recipe)
  • Nunc C96 Maxisorp MicroWell plates (Thermo Fisher Scientific)
  • Multiskan EX Microplate photometer (Thermo Fisher Scientific)
  • VITROS automated chemistry analyzer (Ortho Clinical Diagnostics)
  • Scan Scope digital slide scanner (Aperio Technologies)
  • Image J software (National Institutes of Health, Bethesda, Maryland)
  • DeltaVision Core microscope (Applied Precision)
  • 1‐ml syringe
  • 27‐G needle
  • Metabolic cages
  • Water bottles
  • 5‐ml urine collection tubes
  • Additional reagents and equipment for euthanasia (unit 1.8; Donovan and Brown, ), parenteral injections (unit 1.6; Donovan and Brown, ), blood collection (unit 1.7; Donovan and Brown, ), histology (unit 21.4; Hofman and Taylor, ), and flow cytometric analysis (Coligan et al., ).
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Literature Cited

Literature Cited
  Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M., and Strober, W. 2014. Current Protocols in Immunology. Chapter 5, John Wiley & Sons, Hoboken, NJ.
  Donovan, J. and Brown, P. 2006a. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Donovan, J. and Brown, P. 2006b. Parenteral Injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Donovan, J. and Brown, P. 2006c. Blood Collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9.
  Hofman, F.M. and Taylor, C.R. 2013. Immunohistochemistry. Curr. Protoc. Immunol. 103:21.4.1‐21.4.26.
  Lee, V.W. and Harris, D.C. 2011. Adriamycin nephropathy: A model of focal segmental glomerulosclerosis. Nephrology 16:30‐38.
  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.
  Papeta, N., Zheng, Z., Schon, E.A., Brosel, S., Altintas, M.M., Nasr, S.H., Reiser, J., D'Agati, V.D., and Gharavi, A.G. 2010. Prkdc participates in mitochondrial genome maintenance and prevents Adriamycin‐induced nephropathy in mice. J. Clin. Invest. 120:4055‐4064.
  Polhill, T., Zhang, G.Y., Hu, M., Sawyer, A., Zhou, J.J., Saito, M., Webster, K.E., Wang, Y., Grey, S.T., Sprent, J., Harris, D.C., Alexander, S.I., and Wang, Y.M. 2012. IL‐2/IL‐2Ab complexes induce regulatory T cell expansion and protect against proteinuric CKD. J. Am. Soc. Nephrol. 23:1303‐1308.
  Rangan, G.K., Wang, Y., Tay, Y.C., and Harris, D.C. 1999. Inhibition of nuclear factor‐kappaB activation reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int. 56:118‐134.
  Rangan, G.K., Wang, Y., and Harris, D.C. 2001. Induction of proteinuric chronic glomerular disease in the rat (Rattus norvegicus) by intracardiac injection of doxorubicin hydrochloride. Contemp. Top. Lab. Anim. Sci. 40:44‐49.
  Sun, Y.B., Qu, X., Zhang, X., Caruana, G., Bertram, J.F., and Li, J. 2013. Glomerular endothelial cell injury and damage precedes that of podocytes in adriamycin‐induced nephropathy. PLoS One 8:e55027.
  Wang, Y., Wang, Y.P., Tay, Y.C., and Harris, D.C. 2000. Progressive adriamycin nephropathy in mice: Sequence of histologic and immunohistochemical events. Kidney Int. 58:1797‐1804.
  Wang, Y., Wang, Y.P., Tay, Y.C., and Harris, D.C. 2001. Role of CD8(+) cells in the progression of murine adriamycin nephropathy. Kidney Int. 59:941‐949.
  Wang, Y.M., Zhang, G.Y., Wang, Y., Hu, M., Wu, H., Watson, D., Hori, S., Alexander, I.E., Harris, D.C., and Alexander, S.I. 2006. Foxp3‐transduced polyclonal regulatory T cells protect against chronic renal injury from adriamycin1014. J. Am. Soc. Nephrol. 17:697‐706.
  Zheng, G., Wang, Y., Xiang, S.H., Tay, Y.C., Wu, H., Watson, D., Coombes, J., Rangan, G.K., Alexander, S.I., and Harris, D.C. 2006. DNA vaccination with CCL2 DNA modified by the addition of an adjuvant epitope protects against “nonimmune” toxic renal injury. J. Am. Soc. Nephrol. 17:465‐474.
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