title: Mouse model of proximal tubule endocytic dysfunction.
abstract: BACKGROUND
  Several studies have indicated the central role of the megalin/cubilin multiligand endocytic receptor complex in protein reabsorption in the kidney proximal tubule. However, the poor viability of the existing megalin-deficient mice precludes further studies and comparison of homogeneous groups of mice.
  
  
  METHODS
  Megalin- and/or cubilin-deficient mice were generated using a conditional Cre-loxP system, where the Cre gene is driven by the Wnt4 promoter. Kidney tissues from the mice were analysed for megalin and cubilin expression by quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemistry. Renal albumin uptake was visualized by immunohistochemistry. Twenty-four-hour urine samples were collected in metabolic cages and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. Urinary albumin/creatinine ratios were measured by ELISA and the alkaline picrate method.
  
  
  RESULTS
  The Meg(lox/lox);Cre(+), Cubn(lox/lox);Cre(+) and Meg(lox/lox), Cubn(lox/lox);Cre(+) mice were all viable, fertile and developed normal kidneys. Megalin and/or cubilin expression, assessed by immunohistology and western blotting, was reduced by >89%. Consistent with this observation, the mice excreted megalin and cubilin ligands such as transferrin and albumin in addition to low-molecular weight proteins. We further show that megalin/cubilin double-deficient mice excrete albumin with an average of 1.45 ± 0.54 mg/day, suggesting a very low albumin concentration in the glomerular ultrafiltrate.
  
  
  CONCLUSIONS
  We report here the efficient genetic ablation of megalin, cubilin or both, using a Cre transgene driven by the Wnt4 promoter. The viable megalin/cubilin double-deficient mice now allow for detailed large-scale group analysis, and we anticipate that the mice will be of great value as an animal model for proximal tubulopathies with disrupted endocytosis.
authors.affiliation: Department of Biomedicine, University of Aarhus, Aarhus, Denmark.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Weyer
authors.forename: Tina
authors.initials: T
authors.lastname: Storm
authors.forename: Jingdong
authors.initials: J
authors.lastname: Shan
authors.forename: Seppo
authors.initials: S
authors.lastname: Vainio
authors.forename: Renata
authors.initials: R
authors.lastname: Kozyraki
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Verroust
authors.forename: Erik I
authors.initials: EI
authors.lastname: Christensen
authors.forename: Rikke
authors.initials: R
authors.lastname: Nielsen
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D003404:Creatinine / Q000652:urine; D004195:Disease Models, Animal; D004705:Endocytosis / Q000502:physiology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007124:Immunoenzyme Techniques; D019426:Integrases / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000503:physiopathology*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000502:physiology*; D060528:Wnt4 Protein / Q000235:genetics
chemical_list: D000418:Albumins; D026561:Low Density Lipoprotein Receptor-Related Protein-2; C055127:Lrp2 protein, mouse; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D060528:Wnt4 Protein; C555582:Wnt4 protein, mouse; C485184:enhanced green fluorescent protein; C029486:intrinsic factor-cobalamin receptor; D049452:Green Fluorescent Proteins; D003404:Creatinine; C045073:Cre recombinase; D019426:Integrases