Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity

Abstract

Blessing NW, Blount MA, Sands JM, Martin CF, Klein JD. Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity. Am J Physiol Renal Physiol 295: F1336 -F1341, 2008. First published August 20, 2008 doi:10.1152/ajprenal.90228.2008.-The UT-A1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membraneassociated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 Ϯ 6%; UT-A3 increased 46 Ϯ 13%) or sucrose (UT-A1 increased 81 Ϯ 13%; UT-A3 increased 60 Ϯ 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl-and sucrose-but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity.renal; osmolality; concentrating mechanism; trafficking THE INNER MEDULLA is often hypertonic, especially during antidiuresis, when plasma vasopressin levels are high (12). Vasopressin stimulates urea transport across perfused rat terminal inner medullary collecting ducts (IMCD) (13). Urea transport is also stimulated by hyperosmolarity (resulting from addition of NaCl) in the absence of vasopressin and is further stimulated in the presence of vasopressin (14). While both vasopressin and hyperosmolarity stimulate urea transport, they do so through different second messenger pathways; vasopressin acts by increasing cAMP while hyperosmolarity acts by increasing intracellular calcium but does not increase cAMP (6, 18).UT-A1 is the major urea transport protein expressed in the IMCD (15). Vasopressin increases urea flux in Madin-Darby canine kidney (MDCK) cells that are stably transfected with UT-A1 (4). In rat IMCD suspensions, vasopressin increases both UT-A1 phosphorylation and UT-A1 plasma membrane accumulation (8). Vasopressin stimulation of UT-A1 phosphorylation can be blocked by the protein kinase A (PKA) inh...

Diabetes induced renal urea transport alterations assessed with 3D hyperpolarized13C,15N-Urea

“…Thus, we speculate that this increase is to compensate for the reduced concentrating ability in the diabetic kidney. Likewise, as UT-A2 and UT-B compensate for the action of the each other, the increase observed in UT-B and thereby a decrease in UT-A2 may result from a challenge to restore inner medullary interstitial urea, which is disrupted even in the early stage of diabetes (11,13,15). Although changes are observed both in urea gradient and urea transporter expression, the NGAL marker included in this study indicated that no kidney injury can be seen in the early stages of diabetes.…”

Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion

“…Vasopressin and hyperosmolality have an additive stimulatory effect on urea permeability (11,16,17). Hyperosmolality, similar to vasopressin, increases the phosphorylation and the plasma membrane accumulation of both UT-A1 and UT-A3 (14,26,32,33). However, hyperosmolality and vasopressin signal through different pathways: hyperosmolality via increases in protein kinase Ca and Figure 6.…”

Glycogen Synthase Kinase-3 Modulates Hyperosmotic-Induced Urea Transporter A1 Relocation in the Inner Medullary Collecting Duct Cells

“…Then, cells were treated with hyperosmolar solutions containing 550 or 800 mOsm/kg H 2 O for 1 h. Medium components used in acute adaptation were 250 mOsm/kg H 2 O (125 mmol/l) NaCl, 250 mOsm/kg H 2 O (250 mmol/l) urea, or 125 mOsm/kg H 2 O (62.5 mmol/l) NaCl plus 125 mOsm/kg H 2 O (125 mmol/l) urea, by adding the desired volume of solute stock solutions of 2.5 mol/l NaCl or 5 mol/l urea prepared in iso medium and filter sterilized. For hypertonic stimulation and release experiments, cells were stimulated with 800 mOsm/kg H 2 O NaCl plus urea for 1 h to redistribute UT-A1 and then cells were cultured in iso medium for 2 h to allow recovery [1]. Cells were then harvested after treatment for further analysis.…”

“…Biotinylation of the cell surface was carried out with the Pierce Cell Surface Protein Isolation kit (Pierce) following the manufacturer's instructions [1,12]. At the desired times after treatment, cell surface proteins were labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-biotin; Pierce).…”

“…The cells in the inner medulla are highly specialized and are effectors that not only concentrate the urine but also regulate the widely fluctuating NaCl and urea concentrations [1,2]. A number of studies have reported the accumulation of UT-A1 in the plasma membrane in response to hyperosmolar solutions containing NaCl or sucrose [1] and that hyperosmolality could increase urea permeability in perfused rat terminal IMCDs [3]. However, the mechanisms underlying these effects are not well understood.…”