Mouse Model for Pyelonephritis

André P. Tittel1, Christoph Heuser1, Christian Kurts1

1 Institutes of Molecular Medicine and Experimental Immunology (IMMEI), Rheinische Friedrich‐Wilhelms University, Bonn, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.23
DOI:  10.1002/0471142735.im1523s101
Online Posting Date:  April, 2013
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Abstract

This unit describes a simple way to induce pyelonephritis in female mice using uropathogenic Escherichia coli (UPEC). Methods for culturing and preparing working stocks of UPEC are provided. Protocols to measure the bacterial load in the murine kidney following the establishment of pyelonephritis by determining the bacterial colony forming units (CFU) are also included. This assay can be performed with kidney homogenates if the bacterial load is the sole read‐out or if cellular intactness is not required for subsequent assays, or from kidney single cell suspensions prepared by enzymatic digestion. A support protocol describes methods for isolating cells for flow cytometry, ex vivo culture, and other assays. Curr. Protoc. Immunol. 101:15.23.1‐15.23.9. © 2013 by John Wiley & Sons, Inc.

Keywords: mouse model; pyelonephritis; UPEC

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Pyelonephritis
  • Support Protocol 1: Preparation of the Uropathogenic E. coli (UPEC) Working Stock
  • Support Protocol 2: Determining Kidney Colony Forming Units
  • Support Protocol 3: Determining Phagocytosis of Bacteria by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Induction of Pyelonephritis

  Materials
  • Bacterial working stock (see protocol 2)
  • LB medium (see recipe)
  • 200 mg/ml streptomycin sulfate salt (see recipe)
  • Glycerol
  • Female mice (8 to 12 weeks old)
  • 2,2,2‐Tribromoethanol (see recipe)
  • Instillagel (Farco Pharma)
  • 250‐ml sterile conical flasks without baffle
  • Incubator shaker (New Brunswick Scientific, Innova 4400, 1‐in. orbit)
  • 50‐ml tubes
  • Centrifuge
  • Light microscope
  • 1‐ml syringe
  • Flexible polyethylene catheter (BD Vasculon Plus; 0.6 × 19–mm)
  • Additional reagents and equipment for mouse anesthesia (unit 1.4)

Support Protocol 1: Preparation of the Uropathogenic E. coli (UPEC) Working Stock

  Materials
  • Nitrogen‐stored source stock of uropathogenic E. coli (STEC Center #TW07504)
  • LB medium (see recipe)
  • Streptomycin sulfate salt (see recipe) (200 mg/ml)
  • 100% glycerol (sterile), store for 6 months at 4°C
  • Agar plates (e. g. CPD ID 3 agar plates, Biomerieux)
  • 250‐ml sterile conical flasks without baffle
  • Incubator shaker (1‐in. orbit; Innova 4400, New Brunswick Scientific)
  • Bench‐top centrifuge
  • Sterile 96‐well cell culture plate

Support Protocol 2: Determining Kidney Colony Forming Units

  Materials
  • Mice
  • Phosphate‐buffered saline (PBS), pH 7.4 (ice‐cold and autoclaved; store 1 month at 4°C)
  • 70% ethanol
  • Digestion medium (see recipe)
  • FACS buffer: 0.2% FCS in PBS
  • Red cell removal buffer (see unit 3.1)
  • Agar plates (e. g. CPD ID 3 agar plates, Biomerieux)
  • Scissors, forceps, scalpels
  • 2‐ml sterile tubes
  • ULTRA‐TURRAX T 10 basic (IKA)
  • Sterile 12‐ or 24‐ and 96‐well cell culture plates
  • 37°C incubator/shaker
  • Plunger of 2‐ml syringe
  • 100‐µm nylon mesh
  • 15‐ml polystyrene tubes
  • Centrifuge
  • Additional reagents and equipment for mouse euthanasia (unit 1.4)

Support Protocol 3: Determining Phagocytosis of Bacteria by Flow Cytometry

  Materials
  • Stock of uropathogenic E. coli strain 536‐GFP (Engel et al., )
  • Stock of uropathogenic E. coli strain 536 (STEC center #TW07504)
  • APC Calibrite beads (Becton Dickinson)
  • Fluorochrome‐labeled antibodies for flow cytometry
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Figures

Videos

Literature Cited

Literature Cited
   Beauchamp, D. and Bergeron, M.G. 1999. Pharmacologic basis for the treatment of pyelonephritis. Curr. Infect. Dis. Rep. 1:371‐378.
   Chassin, C., Goujon, J.M., Darche, S., du Merle, L., Bens, M., Cluzeaud, F., Werts, C., Ogier‐Denis, E., Le Bouguenec, C., Buzoni‐Gatel, D., and Vandewalle, A. 2006. Renal collecting duct epithelial cells react to pyelonephritis‐associated Escherichia coli by activating distinct TLR4‐dependent and ‐independent inflammatory pathways. J. Immunol. 177:4773‐4784.
   Engel, D., Dobrindt, U., Tittel, A., Peters, P., Maurer, J., Gutgemann, I., Kaissling, B., Kuziel, W., Jung, S., and Kurts, C. 2006. Tumor necrosis factor alpha‐ and inducible nitric oxide synthase‐producing dendritic cells are rapidly recruited to the bladder in urinary tract infection but are dispensable for bacterial clearance. Infect. Immun. 74:6100‐6107.
   Hagberg, L., Engberg, I., Freter, R., Lam, J., Olling, S., and Svanborg Eden, C. 1983. Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin. Infect. Immun. 40:273‐283.
   Hewitt, I.K., Zucchetta, P., Rigon, L., Maschio, F., Molinari, P.P., Tomasi, L., Toffolo, A., Pavanello, L., Crivellaro, C., Bellato, S., and Montini, G. 2008. Early treatment of acute pyelonephritis in children fails to reduce renal scarring: Data from the Italian Renal Infection Study Trials. Pediatrics 122:486‐490.
   Mulvey, M.A., Lopez‐Boado, Y.S., Wilson, C.L., Roth, R., Parks, W.C., Heuser, J., and Hultgren, S.J. 1998. Induction and evasion of host defenses by type 1‐piliated uropathogenic Escherichia coli. Science 282:1494‐1497.
   Patole, P.S., Schubert, S., Hildinger, K., Khandoga, S., Khandoga, A., Segerer, S., Henger, A., Kretzler, M., Werner, M., Krombach, F., Schlondorff, D., and Anders, H.J. 2005. Toll‐like receptor‐4: Renal cells and bone marrow cells signal for neutrophil recruitment during pyelonephritis. Kidney Int. 68:2582‐2587.
   Roberts, J.A., Domingue, G.J., Martin, L.N., and Kim, J.C. 1981. Immunology of pyelonephritis in the primate model: Live versus heat‐killed bacteria. Kidney Int. 19:297‐305.
   Samuelsson, P., Hang, L., Wullt, B., Irjala, H., and Svanborg, C. 2004. Toll‐like receptor 4 expression and cytokine responses in the human urinary tract mucosa. Infect. Immun. 72:3179‐3186.
   Soos, T.J., Sims, T.N., Barisoni, L., Lin, K., Littman, D.R., Dustin, M.L., and Nelson, P.J. 2006. CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney. Kidney Int. 70:591‐596.
   Svanborg, C., Bergsten, G., Fischer, H., Godaly, G., Gustafsson, M., Karpman, D., Lundstedt, A.C., Ragnarsdottir, B., Svensson, M., and Wullt, B. 2006. Uropathogenic Escherichia coli as a model of host‐parasite interaction. Curr. Opin. Microbiol. 9:33‐39.
   Tardif, M., Beauchamp, D., Bergeron, Y., Lessard, C., Gourde, P., and Bergeron, M.G. 1994. L‐651,392, a potent leukotriene inhibitor, controls inflammatory process in Escherichia coli pyelonephritis. Antimicrob. Agents Chemother. 38:1555‐1560.
   Tittel, A.P., Heuser, C., Ohliger, C., Knolle, P.A., Engel, D.R., and Kurts, C. 2011. Kidney dendritic cells induce innate immunity against bacterial pyelonephritis. J. Am. Soc. Nephrol. 22:1435‐1441.
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