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Uremic cardiomyopathy, characterized by hypertension, cardiac hypertrophy, and fibrosis, is a complication of chronic kidney disease (CKD). Urea transporter (UT) inhibition increases the excretion of water and urea, but the effect on uremic cardiomyopathy has not been studied. We tested UT inhibition by dimethylthiourea (DMTU) in 5/6 nephrectomy mice. This treatment suppressed CKD-induced hypertension and cardiac hypertrophy. In CKD mice, cardiac fibrosis was associated with upregulation of UT and vimentin abundance. Inhibition of UT suppressed vimentin amount. Left ventricular mass index in DMTU-treated CKD was less compared with non-treated CKD mice as measured by echocardiography. Nephrectomy was performed in UT-A1/A3 knockout (UT-KO) to further confirm our finding. UT-A1/A3 deletion attenuates the CKD-induced increase in cardiac fibrosis and hypertension. The amount of α-smooth muscle actin and tgf-β were significantly less in UT-KO with CKD than WT/CKD mice. To study the possibility that UT inhibition could benefit heart, we measured the mRNA of renin and angiotensin-converting enzyme (ACE), and found both were sharply increased in CKD heart; DMTU treatment and UT-KO significantly abolished these increases. Conclusion: Inhibition of UT reduced hypertension, cardiac fibrosis, and improved heart function. These changes are accompanied by inhibition of renin and ACE., (© 2020 Federation of American Societies for Experimental Biology.)

Renal urea handling is central to the urine concentrating mechanism, and as such the ability to image urea transport in the kidney is an important potential imaging biomarker for renal functional assessment. Glucagon levels associated with changes in dietary protein intake have been shown to influence renal urea handling; however, the exact mechanism has still to be fully understood. Here we investigate renal function and osmolite distribution using [ 13 C, 15 N] urea dynamics and 23 Na distribution before and 60 min after glucagon infusion in six female rats. Glucagon infusion increased the renal [ 13 C, 15 N] urea mean transit time by 14%, while no change was seen in the sodium distribution, glomerular filtration rate or oxygen consumption. This change is related to the well-known effect of increased urea excretion associated with glucagon infusion, independent of renal functional effects. This study demonstrates for the first time that hyperpolarized 13 C-urea enables monitoring of renal urinary excretion effects in vivo., (© 2018 John Wiley & Sons, Ltd.)

Purpose of Review: Urea is transported by urea transporter proteins in kidney, erythrocytes, and other tissues. Mice in which different urea transporters have been knocked out have urine-concentrating defects, which has led to the development and testing of urea transporters Slc14A2 (UT-A) and Slc14A1 (UT-B) inhibitors as urearetics. This review summarizes the knowledge gained during the past year on urea transporter regulation and investigations into the clinical potential of urearetics., Recent Findings: UT-A1 undergoes several posttranslational modifications that increase its function by increasing UT-A1 accumulation in the apical plasma membrane. UT-A1 is phosphorylated by protein kinase A, exchange protein activated by cyclic AMP, protein kinase Cα, and AMP-activated protein kinase, all at different serine residues. UT-A1 is also regulated by 14-3-3, which contributes to UT-A1 removal from the membrane. UT-A1 is glycosylated with various glycan moieties in animal models of diabetes mellitus. Transgenic expression of UT-A1 into UT-A1/UT-A3 knockout mice restores urine-concentrating ability. UT-B is present in descending vasa recta and urinary bladder, and is linked to bladder cancer. Inhibitors of UT-A and UT-B have been developed that result in diuresis with fewer abnormalities in serum electrolytes than conventional diuretics., Summary: Urea transporters play critical roles in the urine-concentrating mechanism. Urea transport inhibitors are a promising new class of diuretic agent.

In humans, urea is excreted in sweat, largely through the eccrine sweat gland. The urea concentration in human sweat is elevated when compared to blood urea nitrogen. The sweat urea nitrogen (UN) of patients with end-stage kidney disease (ESRD) is increased when compared with healthy humans. The ability to produce sweat is maintained in the overwhelming majority of ESRD patients. A comprehensive literature review found no reports of sweat UN neither in healthy rodents nor in rodent models of chronic kidney disease (CKD). Therefore, this study measured sweat UN concentrations in healthy and uremic rats. Uninephrectomy followed by renal artery ligation was used to remove 5/6 of renal function. Rats were then fed a high-protein diet to induce uremia. Pilocarpine was used to induce sweating. Sweat droplets were collected under oil. Sweat UN was measured with a urease assay. Serum UN was measured using a fluorescent ortho-pthalaldehyde reaction. Immunohistochemistry (IHC) was accomplished with a horseradish peroxidase and diaminobenzidine technique. Sweat UN in uremic rats was elevated greater than two times compared to healthy pair-fed controls (220 ± 17 and 91 ± 15 mmol/L, respectively). Post hoc analysis showed a significant difference between male and female uremic sweat UN (279 ± 38 and 177 ± 11 mmol/L, respectively.) IHC shows, for the first time, the presence of the urea transporters UT-B and UT-A2 in both healthy and uremic rat cutaneous structures. Future studies will use this model to elucidate how rat sweat UN and other solute excretion is altered by commonly prescribed diuretics., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) have very low levels of urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability., (Copyright © 2016 by the American Society of Nephrology.)

The kidney's ability to concentrate urine is vitally important to our quality of life. In the hypertonic environment of the kidney, urea transporters must be regulated to optimize function. We previously showed that hypertonicity increases urea permeability and that the protein kinase C (PKC) blockers chelerythrine and rottlerin decreased hypertonicity-stimulated urea permeability in rat inner medullary collecting ducts (IMCDs). Because PKCα knockout (PKCα(-/-)) mice have a urine-concentrating defect, we tested the effect of hypertonicity on urea permeability in isolated perfused mouse IMCDs. Increasing the osmolality of perfusate and bath from 290 to 690 mosmol/kgH(2)O did not change urea permeability in PKCα(-/-) mice but significantly increased urea permeability in wild-type mice. To determine whether the response to protein kinase A was also missing in IMCDs of PKCα(-/-) mice, tubules were treated with vasopressin and subsequently with the PKC stimulator phorbol dibutyrate (PDBu). Vasopressin stimulated urea permeability in PKCα(-/-) mice. Like vasopressin, forskolin stimulated urea permeability in PKCα(-/-) mice. We previously showed that, in rats, vasopressin and PDBu have additive stimulatory effects on urea permeability. In contrast, in PKCα(-/-) mice, PDBu did not further increase vasopressin-stimulated urea permeability. Western blot analysis showed that expression of the UT-A1 urea transporter in IMCDs was increased in response to vasopressin in wild-type mice as well as PKCα(-/-) mice. Hypertonicity increased UT-A1 phosphorylation in wild-type mice but not in PKCα(-/-) mice. We conclude that PKCα mediates hypertonicity-stimulated urea transport but is not necessary for vasopressin stimulation of urea permeability in mouse IMCDs.

In the late 1980s, urea permeability measurements produced values that could not be explained by paracellular transport or lipid phase diffusion. The existence of urea transport proteins were thus proposed and less than a decade later, the first urea transporter was cloned. The family of urea transporters has two major subgroups, designated SLC14A1 (or UT-B) and Slc14A2 (or UT-A). UT-B and UT-A gene products are glycoproteins located in various extra-renal tissues however, a majority of the resulting isoforms are found in the kidney. The UT-B (Slc14A1) urea transporter was originally isolated from erythrocytes and two isoforms have been reported. In kidney, UT-B is located primarily in the descending vasa recta. The UT-A (Slc14A2) urea transporter yields six distinct isoforms, of which three are found chiefly in the kidney medulla. UT-A1 and UT-A3 are found in the inner medullary collecting duct (IMCD), while UT-A2 is located in the thin descending limb. These transporters are crucial to the kidney's ability to concentrate urine. The regulation of urea transporter activity in the IMCD involves acute modification through phosphorylation and subsequent movement to the plasma membrane. UT-A1 and UT-A3 accumulate in the plasma membrane in response to stimulation by vasopressin or hypertonicity. Long-term regulation of the urea transporters in the IMCD involves altering protein abundance in response to changes in hydration status, low protein diets, or adrenal steroids. Urea transporters have been studied using animal models of disease including diabetes mellitus, lithium intoxication, hypertension, and nephrotoxic drug responses. Exciting new genetically engineered mouse models are being developed to study these transporters.

Urea transport proteins were initially proposed to exist in the kidney in the late 1980s when studies of urea permeability revealed values in excess of those predicted by simple lipid-phase diffusion and paracellular transport. Less than a decade later, the first urea transporter was cloned. Currently, the SLC14A family of urea transporters contains two major subgroups: SLC14A1, the UT-B urea transporter originally isolated from erythrocytes; and SLC14A2, the UT-A group with six distinct isoforms described to date. In the kidney, UT-A1 and UT-A3 are found in the inner medullary collecting duct; UT-A2 is located in the thin descending limb, and UT-B is located primarily in the descending vasa recta; all are glycoproteins. These transporters are crucial to the kidney's ability to concentrate urine. UT-A1 and UT-A3 are acutely regulated by vasopressin. UT-A1 has also been shown to be regulated by hypertonicity, angiotensin II, and oxytocin. Acute regulation of these transporters is through phosphorylation. Both UT-A1 and UT-A3 rapidly accumulate in the plasma membrane in response to stimulation by vasopressin or hypertonicity. Long-term regulation involves altering protein abundance in response to changes in hydration status, low protein diets, adrenal steroids, sustained diuresis, or antidiuresis. Urea transporters have been studied using animal models of disease including diabetes mellitus, lithium intoxication, hypertension, and nephrotoxic drug responses. Exciting new animal models are being developed to study these transporters and search for active urea transporters. Here we introduce urea and describe the current knowledge of the urea transporter proteins, their regulation, and their role in the kidney., (© 2011 American Physiological Society. Compr Physiol 1:699-729, 2011.)

Terrestrial life would be miserable without the ability to concentrate urine. Production of concentrated urine requires complex interactions among the nephron segments and vasculature in the kidney medulla. In addition to water channels (aquaporins) and sodium transporters, urea transporters are critically important to the theories proposed to explain the physiologic processes occurring when urine is concentrated. Vasopressin (anti-diuretic hormone) is the key hormone regulating the production of concentrated urine. Vasopressin rapidly increases water and urea transport in the terminal inner medullary collecting duct (IMCD). Vasopressin rapidly increases urea permeability in the IMCD through increases in phosphorylation and apical plasma-membrane accumulation of the urea transporter A1 (UT-A1). Vasopressin acts through two cAMP-dependent signaling pathways in the IMCD: protein kinase A and exchange protein activated by cAMP Epac. Protein kinase A phosphorylates UT-A1 at serines 486 and 499. In summary, vasopressin regulates urea transport acutely by increasing UT-A1 phosphorylation and the apical plasma-membrane accumulation of UT-A1 through two cAMP-dependent pathways.

Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its NH(2)-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central part, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these phosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1.

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by (32)P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK-ERK pathway.

In our previous studies of varying osmotic diuresis, UT-A1 urea transporter increased when urine and inner medullary (IM) interstitial urea concentration decreased. The purposes of this study were to examine 1) whether IM interstitial tonicity changes with different urine urea concentrations during osmotic dieresis and 2) whether the same result occurs even if the total urinary solute is decreased. Rats were fed a 4% high-salt diet (HSD) or a 5% high-urea diet (HUD) for 2 wk and compared with the control rats fed a regular diet containing 1% NaCl. The urine urea concentration decreased in HSD but increased in HUD. In the IM, UT-A1 and UT-A3 urea transporters, CLC-K1 chloride channel, and tonicity-enhanced binding protein (TonEBP) transcription factor were all increased in HSD and decreased in HUD. Next, rats were fed an 8% low-protein diet (LPD) or a 0.4% low-salt diet (LSD) to decrease the total urinary solute. Urine urea concentration significantly decreased in LPD but significantly increased in LSD. Rats fed the LPD had increased UT-A1 and UT-A3 in the IM base but decreased in the IM tip, resulting in impaired urine concentrating ability. The LSD rats had decreased UT-A1 and UT-A3 in both portions of the IM. CLC-K1 and TonEBP were unchanged by LPD or LSD. We conclude that changes in CLC-K1, UT-A1, UT-A3, and TonEBP play important roles in the renal response to osmotic diuresis in an attempt to minimize changes in plasma osmolality and maintain water homeostasis.

Trans-epithelial tracer urea flux across Madin-Darby canine kidney (MDCK) cells permanently expressing the urea transporter UT-A1 is stimulated by agents that activate the cAMP signaling pathway, such as vasopressin or forskolin, thus mimicking the activation of urea permeability in the inner medullary collecting duct in the presence of vasopressin. Here, we report that UT-A1-mediated urea flux is also activated two-to-threefold over background by exposing the cells to media containing LiCl. This is in contrast to reports on cortical and medullary collecting duct tubules where acute and chronic exposure to lithium (Li) suppresses the osmotic water permeability, which is also regulated by cAMP levels. The Li concentration dependence of urea flux activation was linear up to 150 mM Li. Li activated only from the basolateral side where its effect was inhibited by amiloride, presumably because Li entered the cells through a basolateral Na-H exchanger. Li and IBMX, which also weakly activated urea flux, greatly augmented each others' stimulatory effect on urea flux. However, cellular cAMP levels did not rise commensurately with urea fluxes, and even though Li augments the activation by forskolin, it greatly inhibits the forskolin-induced formation of cAMP. These results suggest that the effect of Li in this MDCK model of renal cells does not involve cAMP or at least utilizes an additional signaling pathway independent of cAMP.

The kidney responds to high levels of ANG II, as may occur during malignant hypertension, by increasing sodium and water excretion. To study whether kidney medullary transporters contribute to this response, rats were made hypertensive using ANG II. Within 3 days of being given ANG II, systolic blood pressure (BP) was increased (200 mmHg), vs control (130 mmHg), and remained high through day 14. Kidney inner medullary (IM) tip and base and outer medulla were analyzed for transporter protein abundance. There were significant decreases in UT-A1 urea transporter, aquaporin-2 (AQP2) water channel, and NKCC2/BSC1 Na(+)-K(+)-2Cl(-) cotransporter. To determine whether the decreases were a response to hypertension, ANG II, or an ANG II-induced increase in aldosterone, rats were given 1) norepinephrine (to increase BP) and 2) ANG II plus spironolactone (to block the mineralocorticoid receptor). Norepinephrine (7 days) increased BP, urine volume, sodium excretion, and decreased urine osmolality and UT-A1, AQP2, and NKCC2/BSC1 abundances, similar to ANG II. ANG II alone or with spironolactone yielded similar increases in BP, urine volume, and urine osmolality, and decreases in UT-A1 and AQP2 proteins in the IM tip. Plasma vasopressin was unaffected by treatment. Water diuresis did not change UT-A1 but decreased AQP2 and NKCC2/BSC1 abundances. We conclude that decreases in UT-A1, AQP2, and NKCC2/BSC1 proteins may contribute to the diuresis and natriuresis that occur following ANG II or norepinephrine-induced acute hypertension and do not appear to involve ANG II stimulation of aldosterone or thirst.

Transepithelial [(14)C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol.cm(-2).min(-1) in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within approximately 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30-40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V(2) receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to approximately 5 nmol.cm(-2).min(-1). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 microM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 microM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A.

The vasopressin-regulated urea transporter (UT)-A1 is a transmembrane protein with two glycosylated forms of 97 and 117 kDa; both are derived from a single 88-kDa core protein. However, the precise molecular sites and the function for UT-A1 N-glycosylation are not known. In this study, we compared Madin-Darby canine kidney cells stably expressing wild-type (WT) UT-A1 to Madin-Darby canine kidney cell lines stably expressing mutant UT-A1 lacking one (A1m1, A1m2) or both glycosylation sites (m1m2). Site-directed mutagenesis revealed that UT-A1 has two glycosylation sites at Asn-279 and -742. Urea flux is stimulated by 10 nM vasopressin (AVP) or 10 microM forskolin (FSK) in WT cells. In contrast, m1m2 cells have a delayed and significantly reduced maximal urea flux. A 15-min treatment with AVP and FSK significantly increased UT-A1 cell surface expression in WT but not in m1m2 cells, as measured by biotinylation. We confirmed this finding using immunostaining. Membrane fractionation of the plasma membrane, Golgi, and endoplasmic reticulum revealed that AVP or FSK treatment increases UT-A1 abundance in both Golgi and plasma membrane compartments in WT but not in m1m2 cells. Pulse-chase experiments showed that UT-A1 half-life is reduced in m1m2 cells compared with WT cells. Our results suggest that mutation of the N-linked glycosylation sites reduces urea flux by reducing UT-A1 half-life and decreasing its accumulation in the apical plasma membrane. In vivo, inner medullary collecting duct cells may regulate urea uptake by altering UT-A1 glycosylation in response to AVP stimulation.

In normal rats, vasopressin and hyperosmolality enhance urea permeability (P(urea)) in the terminal, but not in the initial inner medullary collecting duct (IMCD), a process thought to occur through the UT-A1 urea transporter. In the terminal IMCD, UT-A1 is detected as 97- and 117-kDa glycoproteins. However, in the initial IMCD, only the 97-kDa form is detected. During streptozotocin-induced diabetes mellitus, UT-A1 protein abundance is increased, and the 117-kDa UT-A1 glycoprotein appears in the initial IMCD. We hypothesize that the 117-kDa glycoprotein mediates the vasopressin- and osmolality-induced changes in P(urea). Thus, in the present study, we measured P(urea) in in vitro perfused initial IMCDs from diabetic rats by imposing a 5 mM bath-to-lumen urea gradient without any osmotic gradient. Basal P(urea) was similar in control vs. diabetic rats (3 +/- 1 vs. 5 +/- 1 x 10(-5) cm/s, n = 4, P = not significant). Vasopressin (10 nM) significantly increased P(urea) to 16 +/- 5 x 10(-5) cm/s (n = 4, P < 0.05) in diabetic but not in control rats. Forskolin (10 microM, adenylyl cyclase activator) also significantly increased P(urea) in diabetic rats. In contrast, increasing osmolality to 690 mosmol/kg H2O did not change P(urea) in diabetic rats. We conclude that initial IMCDs from diabetic rats have vasopressin- and forskolin-, but not hyperosmolality-stimulated P(urea). The appearance of vasopressin-stimulated P(urea) in initial IMCDs correlates with an increase in UT-A1 protein abundance and the appearance of the 117-kDa UT-A1 glycoprotein in this region during diabetes. This suggests that the 117-kDa UT-A1 glycoprotein is necessary for vasopressin-stimulated urea transport.

Production, recycling, and elimination of urea are important to maintain nitrogen balance. Adaptation to varying loads of urea due to different protein intake or in renal failure may involve changes in urea transport and may possibly affect urea transporters. In this study, we examined the expression of the UT-B urea transporter in rats fed a low-protein diet (LPD), a high-protein diet (HPD), and a 20% urea-supplemented diet. In the kidney, UT-B protein abundance increased in the outer medulla of both LPD-fed rats and 20% urea-fed rats, without changes in the inner medulla of either group compared with controls. In HPD-fed rats, UT-B protein decreased significantly in both the outer and inner medulla. We identified expression of UT-B in the rat colon, as a 2-kb mRNA transcript and as an approximately 45-kDa protein, with apical localization in superficial colon epithelial cells. UT-B also is expressed in rat small intestine. In rat colon, UT-B protein abundance was mildly, but significantly, decreased in LPD-fed and 20% urea-fed rats. UT-B abundance also was examined in the colon of 7/8 nephrectomized, uremic rats and in HPD-fed rats and was not significantly different from that in control rats. These findings indicate that UT-B expression is regulated in response to different loads of urea, with a pattern that suggests involvement of tissue-specific regulatory mechanism in kidney and colon.

Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 Omega.cm(2). Transepithelial [(14)C]urea fluxes were measured at 37 degrees C in a HCO(3)(-)/CO(2) buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [(14)C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts.

The UT-A1 urea transporter plays an important role in maintaining the hyperosmolar milieu of the inner medulla. Vasopressin increases urea permeability in rat terminal inner medullary collecting ducts (IMCDs) within 5-10 min. To elucidate the mechanism, IMCD suspensions were radiolabeled with [(32)P]orthophosphate. UT-A1 was immunoprecipitated and analyzed by autoradiogram and Western blot. Both the 97- and 117-kDa UT-A1 proteins were phosphorylated. Vasopressin treatment increased the phosphorylation of both UT-A1 proteins at 2 min, which peaked at 5-10 min and remained elevated for up to 30 min. There was a discernable increase in UT-A1 phosphorylation with 10 pM and a 50% increase with 10-100 nM vasopressin. 1-Desamino-8-D-arginine vasopressin (dDAVP) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) also increased UT-A1 phosphorylation. The vasopressin-stimulated increase in UT-A1 phosphorylation was blocked by H-89 or a specific peptide inhibitor of protein kinase A. Phosphatase inhibitors (okadaic acid, calyculin) increased UT-A1 phosphorylation. We conclude that vasopressin increases UT-A1 phosphorylation via protein kinase A within 2-5 min in rat IMCDs. This suggests that phosphorylation of UT-A1 may be the mechanism by which vasopressin rapidly increases urea permeability in vivo.

We previously showed that the phosphatases PP1/PP2A and PP2B dephosphorylate the water channel, AQP2, suggesting their role in water reabsorption. In this study, we investigated whether protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B or calcineurin), which are present in the inner medullary collecting duct (IMCD), are regulators of urea and water permeability. Inhibition of calcineurin by tacrolimus increased both basal and vasopressin-stimulated osmotic water permeability in perfused rat IMCDs. However, tacrolimus did not affect osmotic water permeability in the presence of aldosterone. Inhibition of PP2A by calyculin increased both basal and vasopressin-stimulated osmotic water permeability, and aldosterone reversed the increase by calyculin. Previous studies showed that adrenomedullin (ADM) activates PP2A and decreases osmotic water permeability. Inhibition of PP2A by calyculin prevented the ADM-induced decrease in water reabsorption. ADM reduced the phosphorylation of AQP2 at serine 269 (pSer269 AQP2). Urea is linked to water reabsorption by building up hyperosmolality in the inner medullary interstitium. Calyculin increased urea permeability and phosphorylated UT-A1. Our results indicate that phosphatases regulate water reabsorption. Aldosterone and adrenomedullin decrease urea or osmotic water permeability by acting through calcineurin and PP2A, respectively. PP2A may regulate water reabsorption by dephosphorylating pSer269, AQP2, and UT-A1. [ABSTRACT FROM AUTHOR]

Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the inner medullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in the IMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases both the phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of a calcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigated whether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effect of PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolality from 290 to 690 mosmol/kgH2O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either 10 μM chelerythrine or 50 μM rottlerin. To determine the potential synergy between vasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated urea permeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeability was reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transport through a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of a cascade of activations remains to be determined. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In our previous studies of varying osmotic diuresis, UT-A1 urea transporter increased when urine and inner medullary (IM) interstitial urea concentration decreased. The purposes of this study were to examine 1) whether IM interstitial tonicity changes with different urine urea concentrations during osmotic dieresis and 2) whether the same result occurs even if the total urinary solute is decreased. Rats were fed a 4% high-salt diet (HSD) or a 5% high-urea diet (HUD) for 2 wk and compared with the control rats fed a regular diet containing 1% NaC1. The urine urea concentration decreased in HSD but increased in HUD. In the IM, UT-Al and UT-A3 urea transporters, CLC-K1 chloride channel, and tonicity-enhanced binding protein (T0nEBP) transcription factor were all increased in HSD and decreased in HUD. Next, rats were fed an 8% low-protein diet (LPD) or a 0.4% low-salt diet (LSD) to decrease the total urinary solute. Urine urea concentration significantly decreased in LPD but significantly increased in LSD. Rats fed the LPD had increased UT-A1 and UT-A3 in the IM base but decreased in the IM tip, resulting in impaired urine concentrating ability. The LSD rats had decreased UT-A1 and UT-A3 in both portions of the IM. CLC-K1 and TonEBP were unchanged by LPD or LSD. We conclude that changes in CLC-K1, UT-A1, UT-A3, and TonEBP play important roles in the renal response to osmotic diuresis in an attempt to minimize changes in plasma osmolality and maintain water homeostasis. [ABSTRACT FROM AUTHOR]

UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT- A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation. [ABSTRACT FROM AUTHOR]

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. Membrane- associated UT-A1 and UT-A3 increased relative to control levels when either NaCI (UT-A1 increased 37 ± 6%; UT-A3 increased 46 ± 13%) or sucrose (UT-A1 increased 81 ± 13%; UT-A3 increased 60 ± 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 NaCI- 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. [ABSTRACT FROM AUTHOR]

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation. [ABSTRACT FROM AUTHOR]

The article presents a scientific research on the tissue distribution of urea transporter-A (UT-A) and urea transporter-B (UT-B) messenger RNA and protein in rats. Researchers were able to identify UT-A and or UT-B messenger RNA and or protein forms in several organs not typically associated with the metabolism of urea.

Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability. [ABSTRACT FROM AUTHOR]

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kid antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of ∼50- and ∼98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of 14C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa. [ABSTRACT FROM AUTHOR]

In rats with streptozotocin-induced diabetes mellitus for 10-20 days, we showed that the abundance of the major medullary transport proteins involved in the urinary concentrating mechanism, urea transporter (UT-A1), aquaporin-2 (AQP2), and the Na+-K+-2Cl- cotransporter (NKCC2/ BSCl), is increased, despite the ongoing osmotic diuresis. To test whether vasopressin is necessary for these diabetes mellitus-induced changes in UT-A1, AQP2, or NKCC2/BSC1, we studied Brattleboro rats because they lack vasopressin. Brattleboro rats were given vasopressin (2.4 µg/day via osmotic minipump) for 5 or 12 days. At 5 days, vasopressin increased AQP2 protein abundance but decreased UT-A1 abundance compared with untreated Brattleboro rats. At 12 days, vasopressin increased the abundance of both UT-A1 and AQP2 proteins but did not alter NKCC2/BSC1. Next, untreated Brattleboro rats were made diabetic for 10 days by injecting them with streptozotocin (40 mg/kg). Diabetes mellitus increased the abundance of AQP2 and NKCC2/BSC1 proteins, but UT-A1 protein abundance did not increase. Third, vasopressin-treated Brattleboro rats were made diabetic with streptozotocin for 10 days. In vasopressin-treated Brattleboro rats, diabetes mellitus increased UT-A1, AQP2, and NKCC2/BSC1 protein abundances. Vasopressin significantly increased UT-A1 phosphorylation in vasopressin-treated diabetic Brattleboro rats but not in the other groups of Brattleboro rats. We conclude that 1) administering vasopressin to Brattleboro rats for 12 days, but not for 5 days, increases UT-A1 protein abundance and 2) vasopressin is necessary for the increase in UT-A1 protein in diabetic rats but is not necessary for the increase in AQP2 or NKCC2 proteins. [ABSTRACT FROM AUTHOR]

Studies the effects of urea transporters on the L-arginine transport. Inhibitory action of urea on L-arginine transport; Presence of urea transporters-B in the endothelial cells; Uremic levels of urea.

Focuses on the effect of vasopressin on the phosphorylation of UT-A1 urea transporter in rat inner medullary collecting ducts through protein kinase A. Time course of vasopressin stimulation of phosphorylation; Dose-response of phosphorylation to concentrations of vasopressin; Regulation of osmotic water permeability.

Discusses a study which evaluated the ability of angiotensin II in regulating urea transport in the inner medullary collecting duct. Methods; Results; Discussion.

Examines the effects of streptozotocin-induced diabetes mellitus on the expression of vasopresin-regulated urea transporter (VRUT) in diabetic rat inner medulla. Higher values of blood glucose and urine osmolality in rats injected with streptozotocin; Measurement of VRUT protein in the inner medullary base of the kidney.

Renal urea handling is central to the urine concentrating mechanism, and as such the ability to image urea transport in the kidney is an important potential imaging biomarker for renal functional assessment. Glucagon levels associated with changes in dietary protein intake have been shown to influence renal urea handling; however, the exact mechanism has still to be fully understood. Here we investigate renal function and osmolite distribution using [13C,15N] urea dynamics and 23Na distribution before and 60 min after glucagon infusion in six female rats. Glucagon infusion increased the renal [13C,15N] urea mean transit time by 14%, while no change was seen in the sodium distribution, glomerular filtration rate or oxygen consumption. This change is related to the well‐known effect of increased urea excretion associated with glucagon infusion, independent of renal functional effects. This study demonstrates for the first time that hyperpolarized 13C‐urea enables monitoring of renal urinary excretion effects in vivo. Hyperpolarized 13C urea MRI during 60 min infusion of glucagon resulted in an increased 13C‐urea renal mean transit time for urea, without changing GFR, renal blood flow or renal oxygen tension. This change is related to the well‐known effect of increased urea excreation associated with the glucagon infusion, independent of renal functional effects. This study demonstrate that hyperpolarized 13C‐urea enables monitoring of renal urinary excretion effects in vivo. [ABSTRACT FROM AUTHOR]

Urea transport by the inner medullary collecting duct (IMCD) is critically important to the urine concentrating mechanism. Transepithelial urea transport requires urea transporters in both apical and basolateral membranes of the IMCD. It is generally agreed that urea moves across the apical membrane by UT-A1, but the identity of the basolateral urea transporter is controversial. One published account localizes UT-A3 at the basolateral membrane in mouse IMCD providing a mechanism for transepithelial urea transport. This study used immunohistochemistry (IHC) with an antibody to the mouse UT-A3 C-terminus. Another report shows UT-A3 localized to the apical membrane in rat IMCD by immunofluorescence (IF) with an antibody to the rat UT-A3 C-terminus. If UT-A3 is not in the basolateral membrane, then the basolateral urea transporter remains unidentified. The goal of this study: directly compare UT-A3 localization in rat and mouse. UT-A3 was identified by western blot using the mouse UT-A3 antibody, and verified using an N-terminal UT-A1 antibody that sees both UT-A1 and UT-A3. IHC analysis revealed that UT-A3, as well as UT-A1, is most abundant in IM tip. Co-immunoprecipitation did not reveal a UT-A1/ UT-A3 interaction. Both IHC and IF analysis of perfused rat and mouse kidneys showed UT-A3 was predominately expressed at the apical plasma membrane. We conclude that UT-A3 primarily localizes to the apical membrane in both rat and mouse IMCD. The mechanistic advantage of UT-A3 in the apical membrane, in addition to UT-A1, remains to be solved. The identity of the basolateral urea transporter remains unknown. [ABSTRACT FROM AUTHOR]

Trafficking of the urea transporter UT-A1 to the plasma membrane is key in vasopressin-regulated urea transport, but the mechanisms remain unclear. Our experiments show UT-A1 involvement with several SNARE-mediated accessory proteins involved in orientation of proteins in the plasma membrane. One group of proteins involved in membrane orientation is the syntaxin family. We recently reported that snapin is important in the interaction of UT-A1 with syntaxin-4 and SNAP23 (t-SNARE). UT-A1-MDCK cells with adenoviral-mediated over-expression of both snapin and syntaxin-4, show that the association of UT-A1 with syntaxin-4 is enhanced snapin. Over-expression of syntaxin-4 alone showed minimal UT-A1/syntaxin-4 binding. Confocal microscopy of UT-A1 and snapin show colocalization in both cytoplasm and plasma membrane. Co-injection of UT-A1 with snapin cRNA (2 ug) in Xenopus oocytes increased urea influx. Co-injection of UT-A1 with t-SNARE without snapin, decreased urea influx. These results suggest that snapin promotes the initial docking of UT-A1 at the plasma membrane to form a SNARE complex. When we treated UT-A1-MDCK cells with vasopressin or forskolin, UT-A1 and snapin were redistributed to the plasma membrane. Collectively, our data support a role for a snapin syntaxin-4/SNAP23 mediated UT-A1 docking complex in vasopressin-regulated trafficking of UT-A1 in UT-A1-MDCK cells. In addition, we verified that the components of the snapin/t-SNARE/UT-A1 complex are present in rat inner medulla, suggesting that this mechanism may be physiologically important. [ABSTRACT FROM AUTHOR]

In order to study the vasopressin regulation of urea transport in the inner medullary collecting duct (IMCD), we have developed the MDCK-UTA1 cell culture model which permits measurements of transepithelial tracer-urea fluxes. This requires one to confirm that the regulation of urea transport in the MDCK-UTA1 cells occurs, as in the IMCD, in the apical membrane. We permeabilized either the apical or basolateral side of the epithelial monolayer using amphotericin. Basolateral amphotericin had no effect on trans-epithelial tracer urea flux in non-stimulated cells (basal flux: 2 nmol cm-2 min-1). In contrast, apical amphotericin greatly increased urea flux to about 20 nmol cm-2 min-1. This indicates that the apical membrane is the side with the limiting urea permeability. In, the presence of forskolin, flux is stimulated approximately 10-fold by activating the urea transporter UT-A1. Again, basolateral amphotericin had no effect on the urea flux whereas apical amphotericin occasionally increased it a bit further. The apical urea permeabilities induced by amphotericin and forskolin were similar in magnitude. To confirm that the basolateral membrane indeed had the high urea permeability, we performed basolateral urea influx experiments. We measured influx rates of 10-20 nmol cm-2 min-1, with near-complete equilibration of basolateral and intracellular urea concentrations in 1-2 min. There was no significant stimulation of basolateral fluxes by forskolin. As MDCK cells do not appear to express native urea transporter, it is not clear which transport pathways might underlie the high basolateral urea permeability. [ABSTRACT FROM AUTHOR]

The 14-3-3 proteins are a group of highly conserved 30-kD regulatory proteins that mainly bind to phosphorylated residues in target proteins. 14-3-3 proteins interact with numerous cellular proteins and influence a large number of cellular processes. UT-A1 urea transporter contains a potential 14-3-3 recognition motif in the large cytoplasmic loop, suggesting a potential role of 14-3-3 in the regulation of UT-A1. To test this model, we examined the expression pattern of seven 14-3-3 protein isoforms in kidney cortex, outer medulla (OM) and inner medulla (IM). Zeta, beta and tau are highly and widely expressed in kidney cortex, OM and IM, as well as in brain, heart and liver. Gamma, eta, and epsilon are found in cortex, OM, and IM, but mainly expressed in IM. There was no detectable sigma in kidney tissue. We then prepared seven 14-3-3 isoform-specific GST fusion proteins for 14-3-3/UT-Al interaction studies. GST pull-down assays with UT-A1-MDCK cell lysate demonstrated that UT-A1 is in a protein complex with selected 14-3-3 isoforms: gamma eta and zeta. R18 peptide, a high-affinity competitive inhibitor of 14-3-3 interactions, prevented UT-A1 from binding to 14-3-3. To examine whether phosphorylation regulates 14-3-3 and UT-A1 binding, UT-A1-MDCK cells were treated with cAMP/adenylyl cyclase stimulator forskolin (10 µM) for 5, 15, and 30 min. Cell lysates were prepared and used for GST 14-3-3 gamma, eta and zeta pull-down assay. Phosphorylation significantly induced UT-A1 binding to 14-3-3. Our study identified 14-3-3 as a novel and potentially important regulator of urea transport activity. [ABSTRACT FROM AUTHOR]

Examines the effect of glycosylation on urea transporters in the renal medullary collecting ducts. Role of UT-A1 in urinary concentrating mechanism; Use of vasopressin in urea transporter regulation; Presence of UT-A1 in the apical plasma membrane.