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Effect of colchicine on redistribution of cell membrane proteins in the rat proximal tubule

Epithelial cells along the mammalian nephron and in other tissues establish, maintain, and modify their plasma membrane composition by the process of membrane recycling (Brown D and Stow JL, Physiol. Rev. 1996, 76:245). In general, the recycling of various membrane components between the plasma membrane and intracellular organelles by means of endo- and exocytosis depends on intact microtubules (MT). However, the details of the recycling mechanism for specific plasma membrane proteins are poorly understood. In the renal proximal tubule (PT) cells, the rate of the MT-dependent recycling of a particular protein may be related to its localization in the specific plasma membrane domain (brush-border vs. basolateral) or to its association with the membrane, e.g. whether a protein is an endo-, transmembrane-, or ecto-protein. Another discriminating factor in this recycling may be the role the specific protein plays in acute or chronic adaptations to the tubular load with the filtered substrate. In this work, we used an MT-depolimerizing agent, colchicine, to study the distribution of Na/K-ATPase [a basolateral (BLM) transmembrane protein], vacuolar proton-ATPase [V-ATPase; a brush-border membrane (BBM) endo-protein], sodium-phosphate co-transporter type 2 (NaPi-2; a BBM transmembrane protein), and cell adhesion molecule CAM-105 (a BBM ecto-protein) in the rat kidney PT cells. Rats were injected i.p. with a single dose of colchicine (0.35 mg/100 g B.M.) or phosphate-buffered saline (controls). Twelve hours later, the animals were anesthetized and kidneys were fixed by perfusion in vivo. An indirect immunofluorescence cytochemistry in 4 m thick cryosections of the kidney cortex was used to label specific membrane proteins in the PT cell plasma membrane domains and intracellular vesicles. A pattern of staining with the monoclonal and polyclonal antibodies to the specific membrane proteins, followed by the fluorescein- or CY3-labeled secondary antibodies, showed in colchicine-treated rats: a) disappearance of MT in the PT cells, b) marked loss of the 31 kDa V-ATPase subunit and NaPi-2 from the BBM, and their redistribution in numerous membrane vesicles randomly scattered in the cell cytoplasm, and c) unchanged distribution of the CAM-105 and Na/K-ATPase in the BBM and BLM, respectively. We conclude that the abundance of V-ATPase and NaPi-2 in the BBM, e.g. transporters whose concentration in the PT cell membrane is known to fastly adapt to acute changes in acid and phosphate loads in the PT, respectively, are very sensitive to MT depolimerization. The slowly-recycling membrane proteins, such as the Na/K-ATPase in the BLM, and the CAM-105 in the BBM, are not significantly affected by MT disruption.

membrane recycling; microtubules; immunocytochemistry; kidney cortex; rat

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