Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension.

The serine protease, furin, is involved in the activation of a number of proteins most notably epithelial sodium channels (ENaC). The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. UT-A1's amino acid sequence has a consensus furin cleavage site (RSKR) in the N-terminal region. Despite the putative cleavage site, we find that UT-A1, either from the cytosolic or cell surface pool, is not cleaved by furin in CHO cells. This result was further confirmed by an inability of furin to cleave in vitro an 35S-labeled UT-A1 or the 126 N-terminal UT-A1 fragment. Functionally, mutation of the furin site (R78A, R81A) does not affect UT-A1 urea transport activity. However, deletion of the 81-aa N-terminal portion does not affect UT-A1 cell surface trafficking, but seriously impair UT-A1 urea transport activity. Our results indicate that UT-A1 maturation and activation does not require furin-dependent cleavage. The N-terminal 81-aa fragment is required for proper UT-A1 urea transport activity, but its effect is not through changing UT-A1 membrane trafficking.

Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.