Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

Abstract

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressinregulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.Rapid, passive urea absorption from the inner medullary collecting duct (IMCD) is responsible for generation of high urea concentrations in the inner medullary interstitium (1) and consequently plays a central role in the urinary concentrating mechanism (2). The rate of absorption is accelerated by vasopressin (3, 4) via increases in intracellular cyclic AMP (5). Physiological studies in isolated perfused tubules have demonstrated that this urea transport pathway is inhibitable by phloretin and structural analogs of urea (6), and is saturable (7), providing strong evidence for the presence of a facilitated urea transporter in IMCD cells (8). Although urea transport across both apical and basolateral plasma membranes of IMCD cells appears to be mediated by phloretin-sensitive urea transporters (6, 9), urea transport across the apical plasma membrane is rate-limiting for overall transepithelial urea transport and is regulated by vasopressin (9). The mechanism by which vasopressin increases urea transport across the apical plasma membrane of the IMCD has not been investigated. In general, there are two possible mechanisms. (i) As has been demonstrated for the vasopressin-regulated water channel (10-13), the urea permeability of the apical plasma membrane may be increased as a result ofvasopressin-stimulated insertion of urea-transporter-containing vesicles into the apical p...

Aquaporin-2 and urea transporter-A1 are up-regulated in rats with Type I diabetes mellitus

“…In these two zones, urea transporters are not expressed homogeneously. In IS, UT-A2 has been localized in the deep but not in the superficial half of thin descending limb (TDL) of short loops of Henle [14,16,18]. In IM, facilitated urea transport has been shown in the terminal but not in the initial inner medullary collecting duct (CD) (IMCD) [19] and UT-A1 mRNA have been localized in IM tip but not in IM base [18].…”

“…Both UT-A1 and UT-A2 are expressed exclusively in the kidney and are encoded by differential transcription from the same gene. Both UT-A2 and UT-B1 are present on the luminal and the basolateral membranes along a limited length of thin descending limbs (TDL) for UT-A2 [16] and in the endothelium of descending vasa recta along most of their length for UT-B1 [17].…”

Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct

“…Several UT-A proteins are derived from the UT-A gene by the process of alternative splicing, including UT-A1 and UT-A3, expressed in the IMCD (12,13), UT-A2, expressed in the thin descending limb of Henle (14), and UT-A5, expressed in testis (15). The UT-B gene encodes only a single protein, and recently UT-B-deficient mice, which manifest a defect in the urinary concentrating mechanism, have been generated (16).…”

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray,Dasyatis sabina

“…Although we could not identify functional differences between strUT-1 and strUT-2, the finding that the expression of only strUT-2c was lower in rays exposed to a 50% reduction in salinity (discussed below) indicates that the two transporter isoforms could play different roles in tubular reabsorption of urea. Different roles of strUT-1 and strUT-2 in renal urea reabsorption might arise from expression in different nephron segments, which is the case for various isoforms of the UT-A transporters within the mammalian kidney (25,38) or trafficking to different membrane domains in polarized epithelia. Further, similar regulatory mechanisms could contribute to the selective expression of one or more of the 3ЈUTR variants of the strUT-1 and strUT-2 isoforms at different sites along the nephron.…”