Journal
NEPHROLOGY DIALYSIS TRANSPLANTATION
Volume 26, Issue 11, Pages 3446-3451Publisher
OXFORD UNIV PRESS
DOI: 10.1093/ndt/gfr525
Keywords
albuminuria; Cre recombinase; endocytosis; proximal tubule; transgenic mouse
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Funding
- Lundbeck Foundation
- Danish Medical Research Council
- Novo Nordisk Foundation
- European Commission EUNEFRON [201590]
- Danish Kidney Association
- Aase and Ejnar Danielsens Foundation
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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.
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