title: Dynamin2, clathrin, and lipid rafts mediate endocytosis of the apical Na/K/2Cl cotransporter NKCC2 in thick ascending limbs.
abstract: Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis.
authors.affiliation: Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Wayne State University, Detroit, Michigan 48202, USA.
authors.forename: Gustavo R
authors.initials: GR
authors.lastname: Ares
authors.forename: Pablo A
authors.initials: PA
authors.lastname: Ortiz
mesh_terms: D000818:Animals; D015153:Blotting, Western; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D033922:Clathrin Heavy Chains / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034285:Dynamin II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D006835:Hydrazones / Q000494:pharmacology; D008138:Loop of Henle / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1
chemical_list: D051242:Caveolin 1; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D033922:Clathrin Heavy Chains; D034285:Dynamin II; D002746:Chlorpromazine