Cubilin is essential for albumin reabsorption in the renal proximal tubule

Abstract

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.

Figure 1.

Cubilin and megalin inactivation. (A) The Cubn gene was targeted by homologous recombination using a construct containing three loxP sites (red) inserted into intron 13 and intron 14 flanking exon 14 and a hygromycin resistance cassette (blue) in intron 14. Homologous recombination and subsequent Cre recombinase excision generate a mutant allele that lacks exon 14 and an additional 1 kb of upstream and downstream genomic DNA. (B) Total kidney extracts separated on SDS-PAGE (5 to 16% gradient) were stained with Coomassie Blue or analyzed by Western blotting with anti-cubilin antibodies. Note that there is no detectable cubilin in mutant mice. (C through F) Immunohistochemistry of renal cortex of cubilin-deficient mice tested for cubilin (in red, C and E) or megalin (in green, D and F). (G and H) Immunohistochemistry of renal cortex of megalin-deficient mice tested for cubilin (in red, G) or megalin (in green, H). Bar: 100 μm in C and D; 50 μm in E through H.

Figure 2.

Altered expression of amnionless in cubilin-deficient mice. Immunohistochemical detection of amnionless (in red, left panel) and cubilin (in green, right panel) in mosaic tubules of cubilin-deficient mice. The arrowhead points to coexpression of cubilin and amnionless in cubilin-expressing cells. The arrows point to intracellular, perinuclear expression of amnionless in some cells lacking cubilin. In many cells (*) amnionless is not detectable in the absence of cubilin. Bar, 50 μm.

Figure 3.

Uptake of intrinsic factor-cobalamin complexes in cubilin- or megalin-deficient mice. (A) Immunochemical staining for intrinsic factor (in green) in the kidney cortex of cubilin-deficient mice. Note the lack of uptake. (B and C) Double labeling for IF (B in green) or megalin (C in red) in mosaic tubules from the renal cortex of megalin-deficient mice. Uptake of IF is detectable in megalin-deficient cells (arrows) but dramatically increased in cells expressing megalin. The mice were sacrificed 30 minutes after intravenous injection of IF-cobalamin complexes. Bar, 50 μm.

Figure 4.

Urinary proteins in cubilin mutants. (A) Comparison of albumin excretion (μg/18 hours) in control and cubilin-deficient mice. (B) Profile of urinary proteins in control mice and cubilin-deficient, megalin-deficient, or cubilin- and megalin-deficient mice. Equivalent amounts of urine (normalized as in A) were subjected to SDS-PAGE and stained with Coomassie Blue. (C) Identification by Western blotting of RBP, DBP, cathepsin B, transferrin, and CC16 in the urine of cubilin, megalin, or cubilin- and megalin-deficient mice.

Figure 5.

Decreased tubular reabsorption of albumin in cubilin- or megalin-deficient mice. Immunohistochemical detection of mouse albumin (in red, B and D) and megalin (in green, A) or cubilin (in green, C) in mosaic tubules of the kidney cortex from megalin-deficient (A and B) or cubilin-deficient (C and D) mice. Albumin-containing vesicles cannot be identified in cells lacking either megalin or cubilin. Bar, 50 μm.

Figure 6.

Tubular reabsorption of apoA-I in cubilin- or megalin-deficient mice. (A) Immunohistochemical detection of apoA-I in cubilin-deficient mice. Cubilin was not detectable in the tissue. (B through D) Detection of apoA-I (in red, C) and megalin (in green, B) in mosaic tubules from the renal cortex of megalin-deficient mice. (D) Merge. Uptake of apoA-I is detectable in megalin-deficient cells (arrows) but dramatically increased in cells expressing megalin. Mice were sacrificed 30 minutes after intravenous injection of labeled apoA-I. Bar, 50 μm.