Renal Na⁺-K⁺-Cl⁻cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent

Abstract

- ClϪ cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent. Am J Physiol Renal Physiol 295: F789 -F802, 2008. First published June 25, 2008 doi:10.1152/ajprenal.90227.2008.-Apical bumetanide-sensitive NaϪ cotransporter (NKCC2), the kidneyspecific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40 -70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na ϩ -Cl Ϫ cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86 Rb ϩ influx in Xenopus laevis oocytes was markedly reduced by methyl-␤-cyclodextrin (M␤CD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/M␤CD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.

Short-term stimulation of the thiazide-sensitive Na+-Cl−cotransporter by vasopressin involves phosphorylation and membrane translocation

“…In TAL, short-term administration of vasopressin induces significant trafficking of NKCC2 to the luminal membrane in mouse (13) and rat kidney (28,44), which agrees with the significant expression of V 2 R at this site. Assuming that similar changes occur in DCT, the intracellular distribution of NCC was analyzed in DI rats receiving dDAVP (1 ng/g body wt for 30 min) or vehicle.…”

“…The capacity of AVP to increase Na ϩ reabsorption in acute and chronic settings using tissues (8), isolated perfused tubules (15), and cell lines (44) has been appreciated for a long time. Although TAL and collecting ducts are established effector sites in this respect (2, 16), a role for DCT has not been clearly determined so far.…”

“…SPAK and oxidative stress response kinase were found to interact with the phosphoacceptor site of NKCC isoforms (7,27,32,34). Apical trafficking and surface expression of NKCC2 have further been established upon application of AVP (13,25,44), and regulatory domains of NKCC2 have been identified (4,47).…”

“…In TAL, we and others have clearly shown that the V 2 R-mediated activation of NKCC2 is triggered by cAMP and protein kinase A (PKA) (44). One may assume that this applies for the DCT as well, but compelling evidence for the role of cAMP/PKA is still scarce (14), probably because of the difficulty to obtain a suitable cell model for the DCT.…”

Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane

“…M␤CD was used to deplete plasma membrane cholesterol in X. laevis oocytes (32). After 3 days of cRNA microinjection, oocytes were washed with uptake solution and incubated with 10 mM M␤CD (Sigma) in the same solution for 2 h. Cells were then used for urea uptake assay and protein expression as described above.…”