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.
Highlights
Apical NKCC2 mediates NaCl reabsorption by the thick ascending limb
We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native thick ascending limb (THAL); the pathways involved in NKCC2 endocytosis are unknown
Dynamin-dependent NKCC2 Endocytosis in THALs—We previously found that NKCC2 undergoes constitutive endocytosis in THALs, the proteins involved are unknown
Summary
Results: Inhibition of dynamin-2, clathrin and lipid raft-mediated endocytosis blunted NKCC2 endocytosis, increasing steadystate surface NKCC2 levels. 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). Because several apical transporters such as NHE3 [24], ENaC [25, 26], and aquaporin-2 (AQP-2) [27] are known to be internalized via a dynamin2-dependent process in cultured epithelial cells, it is possible that NKCC2 is internalized via dynamin-2 in THALs. Our earlier study showed that depletion of membrane cholesterol with methyl--cyclodextrin (MCD) inhibited NKCC2 internalization and increased steady-state surface NKCC2 in THALs [16]. We tested the hypothesis that surface NKCC2 undergoes endocytosis via a dynamin-2, clathrin and lipid raft-dependent manner in native THALs
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