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Defining the pathway of megalin trafficking in the proximal tubule
Author(s) -
Shipman Katherine,
Long Kimberly,
Weisz Ora
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02856
Subject(s) - endocytic cycle , endocytosis , microbiology and biotechnology , receptor , chemistry , endosome , clathrin , compartment (ship) , transport protein , biology , biochemistry , oceanography , geology
The polarized epithelial cells that comprise the proximal tubule (PT) have a specialized apical endocytic pathway that allows for the high‐capacity endocytosis necessary to recover essential nutrients and to maintain a protein‐free urine. Impairments in this pathway result in tubular proteinuria, which can worsen to cause more severe kidney disease. Megalin and cubilin, multi‐ligand receptors at the apical surface of the epithelial cells, bind proteins in the ultrafiltrate and internalize them via receptor‐mediated endocytosis. Ligands are sorted from receptors in endocytic compartments, and the receptors are recycled back to the surface. The molecular identities of the compartments involved in sorting and recycling in PT cells and the kinetics of megalin trafficking through them are unknown. Understanding megalin's endocytic trafficking itinerary and the key regulatory steps in this pathway is important for discerning the basis of proteinuric diseases and devising therapies. To address this, we identified endocytic compartment markers in a previously developed opossum kidney (OK) cell culture model that recapitulates morphologic and functional features of the PT in vivo . The fraction of total megalin colocalizing with each compartment marker was quantified by Mander's coefficient from deconvolved confocal images. Finally, megalin trafficking was quantified to assess endocytic kinetics and half‐life. Our combined data using surface biotinylation and quantitative imaging revealed that only a small fraction of total megalin is present at the apical surface at steady state. Moreover, megalin is rapidly internalized from the surface and is localized primarily in Rab11a‐positive compartments at steady state. These data suggest that recycling is the rate‐limiting step in regulating the fraction of total megalin available at the surface. Together, our results provide valuable insight into the spatial organization of the endolysomal system and the itinerary and regulation of megalin traffic in the PT. Our biochemical and quantitative colocalization data will be used to construct and refine a kinetic model of megalin trafficking in the PT that can be used to identify how disruptions in the endocytic pathway cause proteinuric disease.