Animal Models of IgA Nephropathy

Steven N. Emancipator1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.11
DOI:  10.1002/0471142735.im1511s29
Online Posting Date:  May, 2001
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Abstract

IgA nephropathy (IgAN) is a form of immune complex glomerulonephritis that occurs spontaneously in humans. This unit describes the induction of active disease in inbred mice, utilizing inert proteins or a common viral pathogen as the inciting antigen. An alternate protocol is offered for the induction of disease in rats by noninfectious protein antigens. Support protocols are presented for the evaluation of the extent of disease, for preparation of infectious and inactivated suspensions of viral stock, and for quantification of the virus.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of IgAN in Mice Using Xenogeneic Inert Proteins
  • Alternate Protocol 1: Induction of IgAN in Rats Using Xenogeneic Inert Proteins
  • Basic Protocol 2: Induction of IgAN in Mice Using Sendai Virus
  • Support Protocol 1: Evaluation of IgAN
  • Support Protocol 2: Preparation of Infectious Sendai Virus Stock
  • Support Protocol 3: Preparation of Inactivated Sendai Virus Stock
  • Support Protocol 4: Quantification of Sendai Virus
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of IgAN in Mice Using Xenogeneic Inert Proteins

  Materials
  • Female BALB/c mice (Table 15.11.1), 4 to 6 weeks old (20 g)
  • Clidox disinfectant, prepared fresh according to manufacturer's directions (Pharmacal Biotech)
  • Protein antigen to induce IgAN: purified water‐soluble protein derived from a nonmurine species (commercial preparations are convenient, but suitable proteins can also be prepared in the laboratory at the investigator's discretion)
  • PBS ( appendix 2A, but adjust to pH 7.5)
  • 6 and 6.6 mM HCl, pH 3.5, prepared fresh from concentrated HCl and tap water
  • Static microisolator cages for mice (unit 1.2), autoclaved, with:
    • Mouse food (Purina Mills or equivalent), autoclaved
    • Rodent bedding, autoclaved
    • 50‐ml water bottles, autoclaved
  • Sterile screw‐cap tubes
  • Acrylic mouse restrainer (Tailveiner, Harvard Instruments, or equivalent)
  • Infrared heat lamp and adjustable stand
  • Sterile 27‐G × 1‐in. (2.54‐cm) needles (Becton Dickinson or equivalent)
  • Sterile plastic tuberculin syringes (Becton Dickinson or equivalent)
  • Metabolic cages for mice
  • 15‐ml calibrated, conical urine collection tubes
  • Mineral oil
  • Additional reagents and equipment for determining protein concentration (e.g., A 280 absorbance) and for urinalysis (see protocol 4)
    Table 5.1.1   Materials   Suitability of Various Murine Strains for Induction of IgAN a   Suitability of Various Murine Strains for Induction of IgAN

    Strain, species IgAN elicited by IgAN not elicited by Sources b References
    A/J, mouse protocol 3 c Harlan, Jackson Jessen et al. ( ) and author's unpub.observ.
    BALB/c, mouse protocol 1, protocol 3 d Charles River, Harlan, Jackson, Taconic Emancipator et al. ( , ), Gesualdo et al. ( ), and author's unpub. observ.
    C3HeB, mouse protocol 3 protocol 1 Charles River, Harlan, Jackson, Taconic Genin et al. ( ) and author's unpub. observ.
    C3H/HeJ, mouse protocol 1 Charles River, Harlan, Jackson, Taconic Genin et al., ( )
    C57BL/6, mouse protocol 3 Charles River, Harlan, Jackson, Taconic Jessen et al. ( ) and author's unpub. observ.
    CAF1, mouse protocol 1 Harlan, Jackson Emancipator et al. ( )
    Lewis, rat protocol 2 Charles River, Harlan Gesualdo et al. ( )
    Wistar, rat protocol 2 Charles River, Harlan Gesualdo et al. ( )
    Fischer, rat protocol 2 Charles River, Harlan, Taconic Gesualdo et al. ( )
    Sprague‐Dawley, rat protocol 2 Charles River, Harlan, Taconic Gesualdo et al. ( )

     aExperience with each strain within the author's laboratory and/or by published account indicated for protocols that elicit IgA‐predominant glomerular immune deposits associated with at least microhematuria as an indicator of glomerular dysfunction, as well as for protocols that do not elicit these defining features of IgAN.
     bAbbreviations are for Charles River Labs, Harlan Bioproducts for Science, Jackson Labs, and Taconic Farms.
     cAlthough this strain develops a clear and significant glomerulonephritis, the humoral immune response to immunization via protocol 3 is not IgA‐predominant and the glomerular deposits are not rich in IgA.
     dIn addition to the development of IgAN in response to either protocol 1 or protocol 32, the BALB/c strain has also been successfully employed in passive IgAN induced by injection of preformed IgA immune complexes (Rifai et al., ; Emancipator et al., ).

Alternate Protocol 1: Induction of IgAN in Rats Using Xenogeneic Inert Proteins

  • Female Lewis or Wistar rats (Table 15.11.1), 4 to 6 weeks old (150 g)
  • Metabolic and housing cages for rats
  • Clean 20 × 20–cm (or larger) towels

Basic Protocol 2: Induction of IgAN in Mice Using Sendai Virus

  Materials
  • Female C3HeB/seJ mice (Table 15.11.1), 4 to 6 weeks old (20 g)
  • Clidox disinfectant, prepared fresh according to manufacturer's directions (Pharmacal Biotech)
  • recipe1 mg/ml inactivated Sendai virus/adjuvant suspension (see recipe)
  • 5 × 109 pfu/ml infectious Sendai virus stock in PBS (see protocol 5)
  • Static microisolator cages for mice (unit 1.2), autoclaved, with:
    •  Mouse food (Purina Mills or equivalent), autoclaved
    •  Rodent bedding, autoclaved
    •  50‐ml water bottles, autoclaved, containing 6 mM HCl in tap water
  • 1.70‐mm‐o.d., 1.19‐mm‐i.d. polyethylene tubing (Fisher or equivalent)
  • Sterile 18‐G × 1‐in. (2.54‐cm) needles (Becton Dickinson or equivalent)
  • Sterile 1‐ml plastic tuberculin syringes (Becton Dickinson or equivalent)
  • Metabolic cages for mice
  • 15‐ml calibrated, conical urine collection tubes
  • Mineral oil

Support Protocol 1: Evaluation of IgAN

  Materials
  • Urine samples to be measured (see protocol 1Basic Protocols 1 and protocol 32; see protocol 2)
  • 2‐methylbutane (isopentane)
  • Liquid nitrogen
  • Cryostat embedding medium (e.g., OCT compound; Fisher)
  • Fluorophore‐conjugated antisera (Organon Teknika Cappel; Southern Biotechnology): specific for mouse or rat IgA, IgG, IgM, and C3, and for the antigen selected for immunization (i.e., anti–inert protein or anti–Sendai virus antisera)
  • 50% (v/v) glycerol in PBS ( appendix 2A, but adjust to pH 7.5)
  • Urine dipsticks for hematuria (Ames Hemastix or equivalent) and proteinuria (Ames Albustix or equivalent) or a combination of the two (Ames Multistix or equivalent)
  • Microscope slides
  • Coverslips
  • Microscope with phase‐contrast optics (preferred), 40× objective, and epifluorescence filters
  • Surgical tools
  • Dewar's flask large enough to contain a 50‐ml beaker
  • Cryostat microtome with metal block holder and knife, chilled to −20°C
  • Additional reagents and equipment for euthanasia (unit 1.8)

Support Protocol 2: Preparation of Infectious Sendai Virus Stock

  Materials
  • Specific‐pathogen‐free embryonated chicken eggs (SPAFAS)
  • ∼109 pfu/ml Sendai virus, strain 52 (ATCC #VR 105)
  • PBS ( appendix 2A, but adjust to pH 7.5), ice cold
  • 70% (v/v) ethanol
  • Tincture of iodine
  • Duro cement
  • 0.5 M EDTA
  • 1 mM EDTA in PBS
  • Egg incubator fitted with wet and dry thermometers, preequilibrated for ≥24 hr to 85° to 87°F (wet thermometer), 99°F (dry thermometer), and ∼60% humidity
  • Bright light or fiberoptic dissecting microscope illuminator
  • Sterile dissecting scissors, forceps
  • Sterile 16‐G, 21‐G, and 25‐G needles (sterile pins can be used in place of 21‐G needles)
  • Sterile disposable tuberculin syringes and 60‐ml syringes
  • Sterile 100‐mm petri dishes
  • 250‐ml sterile screw‐cap centrifuge bottles
  • Super‐speed centrifuge (Sorvall RT‐5C and GSA rotor, or equivalent)
  • Additional reagents and equipment for quantifying virus (see protocol 7)

Support Protocol 3: Preparation of Inactivated Sendai Virus Stock

  • Infectious Sendai virus stock (see protocol 5)
  • β‐propiolactone (e.g., Sigma)

Support Protocol 4: Quantification of Sendai Virus

  Materials
  • Trachea or preweighted lung tissue freshly dissected upon sacrifice of challenged mice (optional)
  • Sterile sand (optional)
  • 1× and 2× medium 199 (e.g., Bio‐Whittaker)
  • 29.2 g/liter L‐glutamine
  • 50 g/liter gentamicin
  • Infectious Sendai virus stock (optional; see protocol 5)
  • Sterile HBSS ( appendix 2A), with Ca+2 and Mg2+
  • Confluent monolayers of LL‐CMK 2 cells(ATCC #18105) maintained in medium 199 supplemented with 5% (v/v) FBS ( appendix 2A), 2 mM L‐glutamine, and 20 µg/ml gentamycin, in 35‐mm2 wells (i.e., 6‐well plates)
  • Agar
  • Tissue culture–grade trypsin
  • Dulbecco's PBS (DPBS) with Ca2+ and Mg+2(Bio‐Whittaker)
  • recipe0.1% (v/v) washed guinea pig erythrocytes (see recipe)
  • Sterile mortar and pestle (optional)
  • Water bath at 42°C
  • Magnifying lens (optional)
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Figures

Videos

Literature Cited

Literature Cited
   de Fijter, J.W., Eijgenraam, J.W., Braam, C.A., Holmgren, J., Daha, M.R., van Es, L.A., and van den Wall Bake, A.W. 1996. Deficient IgA1 immune response to nasal cholera toxin subunit B in primary IgA nephropathy. Kidney Int. 50:952‐961.
   Emancipator, S.N., Gallo, G.R., and Lamm, M.E. 1983. Experimental IgA nephropathy induced by oral immunization. J. Exp. Med. 157:572‐582.
   Emancipator, S.N., Ovary, Z., and Lamm, M.E. 1987. The role of mesangial complement in the hematuria of experimental IgA nephropathy. Lab. Invest. 57:269‐276.
   Emancipator, S.N., Chintalacharuvu, S.R., Bagheri, N., and Scivittaro, V. 1997. Animal models of IgA nephropathy: Formulating therapeutic strategies. Nephrology 3:45‐50.
   Emancipator, S.N., Mestecky, J., and Lamm, M.E. 1999. IgA nephropathy and related diseases. In Mucosal Immunology, 2nd ed. (P.L. Ogra, J. Mestecky, M.E. Lamm, W. Strober, J. Bienenstock, and J. McGhee eds.) pp. 1365‐1380. Academic Press, New York.
   Genin, C., Laurent, B., Sabatier, J.C., Colon, S., and Berthoux, F.C. 1986. IgA mesangial deposits in C3H/HeJ mice after oral immunization with ferritin or bovine serum albumin. Clin. Exp. Immunol. 63:385‐394.
   Gesualdo, L., Lamm, M.E., and Emancipator, S.N. 1990. Defective oral tolerance promotes nephritogenesis in experimental IgA nephropathy induced by oral immunization. J. Immunol. 145:3684‐3691.
   Gesualdo, L., Pinzani, M., Floriano, J.J., Hassan, M.O., Nagy, N.U., Schena, F.P., Emancipator, S.N., and Abbound, H.E. 1991. Platelet‐derived growth factor expression in mesangial proliferative glomerulonephritis. Lab. Invest. 65:160‐167.
   Gesualdo, L., Emancipator, S.N., Kesselheim, C., and Lamm, M.E. 1992. Glomerular hemodynamics and eicosanoid synthesis in a rat model of IgA nephropathy. Kidney Int. 42:106‐114.
   Isaacs, K.L. and Miller, F. 1982. Role of antigen size and charge in immune complex glomerulonephritis. I. Active induction of disease with dextran and its derivatives. Lab. Invest. 47:198‐205.
   Jessen, R.H., Emancipator, S.N., Jacobs, G.H., and Nedrud, J.G. 1992. Experimental IgA‐IgG nephropathy induced by a viral respiratory pathogen: Dependence on antigen form and immune status. Lab. Invest. 67:379‐386.
   Rifai, A. and Mannik, M. 1984. Clearance of circulating IgA immune complexes is mediated by a specific receptor on Kupffer Cells in mice. J. Exp. Med. 160:125‐137.
   Rifai, A., Small, P.A. Jr., Teague, P.O., and Ayoub, E.M. 1979. Experimental IgA nephropathy. J. Exp. Med. 150:1161‐1173.
Key References
   Emancipator, S.N. 1998. IgA nephropathy, and Henoch‐Schönlein syndrome. In Heptinstall's Pathology of the Kidney, 5th ed. (J.C. Jennette, J.L. Olson, M.M. Schwarts, and F.G. Silva, eds.) pp. 479‐540. Lippincott‐Raven, Philadelphia.
  This is an extensive and comprehensive overview of IgAN in patients, with pathogenesis and pathophysiology sections.
   Emancipator et al., 1997. See above.
  This is a synopsis of current activity and past accomplishments in experimental IgAN.
   Emancipator et al., 1999. See above.
  This is an in‐depth analysis of immune dysfunction in IgAN.
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