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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.

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 fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-β, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-β, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson's trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO.

Aldosterone indirectly regulates water reabsorption in the distal tubule by regulating sodium reabsorption. However, the direct effect of aldosterone on vasopressin-regulated water and urea permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether aldosterone regulates osmotic water permeability in isolated perfused rat IMCDs. Adding aldosterone (500 nM) to the bath significantly decreased osmotic water permeability in the presence of vasopressin (50 pM) in both male and female rat IMCDs. Aldosterone significantly decreased aquaporin-2 (AQP2) phosphorylation at S256 but did not change it at S261. Previous studies show that aldosterone can act both genomically and non-genomically. We tested the mechanism by which aldosterone attenuates osmotic water permeability. Blockade of gene transcription with actinomycin D did not reverse aldosterone-attenuated osmotic water permeability. In addition to AQP2, the urea transporter UT-A1 contributes to vasopressin-regulated urine concentrating ability. We tested aldosterone-regulated urea permeability in vasopressin-treated IMCDs. Blockade of gene transcription did not reverse aldosterone-attenuated urea permeability. In conclusion, aldosterone directly regulates water reabsorption through a non-genomic mechanism. Aldosterone-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. There may be a sex difference apparent in the inhibitory effect of aldosterone on water reabsorption in the inner medullary collecting duct. This study is the first to show a direct effect of aldosterone to inhibit vasopressin-stimulated osmotic water permeability and urea permeability in perfused rat IMCDs.

Background: Urea transporters (UTs) are important in urine concentration and in urea recycling, and UT-B has been implicated in both. In kidney, UT-B was originally localized to outer medullary descending vasa recta, and more recently detected in inner medullary descending vasa recta. Endogenously produced microRNAs (miRs) bind to the 3'UTR of genes and generally inhibit their translation, thus playing a pivotal role gene regulation., Methods: Mice were dehydrated for 24 hours then sacrificed. Inner and outer medullas were analyzed by polymerase chain reaction (PCR) and quantitative PCR for miRNA expression and analyzed by western blotting for protein abundance., Results: MiRNA sequencing analysis of mouse inner medullas showed a 40% increase in miRNA-200c in dehydrated mice compared with controls. An in silico analysis of the targets for miR-200c revealed that miRNA-200c could directly target the gene for UT-B. PCR confirmed that miR-200c is up-regulated in the inner medullas of dehydrated mice while western blot showed that UT-B protein abundance was down-regulated in the same portion of the kidney. However, in the outer medulla, miR-200c was reduced and UT-B protein was increased in dehydrated mice., Conclusions: This is the first indication that UT-B protein and miR-200c may each be differentially regulated by dehydration within the kidney outer and inner medulla. The inverse correlation between the direction of change in miR-200c and UT-B protein abundance in both the inner and outer medulla suggests that miR-200c may be associated with the change in UT-B protein in these 2 portions of the kidney medulla., Competing Interests: No conflicts of interest are declared by the authors., (Copyright © 2016 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

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.)

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations., (Copyright © 2016 the American 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.)

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane., (Copyright © 2015 the American Physiological Society.)

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

The urea transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC-α regulates UT-A1 urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC-α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC-α-deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC-α-induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions., (Copyright © 2015 by the American Society of Nephrology.)

UT-A and UT-B families of urea transporters consist of multiple isoforms that are subject to regulation of both acutely and by long-term measures. This chapter provides a brief overview of the expression of the urea transporter forms and their locations in the kidney. Rapid regulation of UT-A1 results from the combination of phosphorylation and membrane accumulation. Phosphorylation of UT-A1 has been linked to vasopressin and hyperosmolality, although through different kinases. Other acute influences on urea transporter activity are ubiquitination and glycosylation, both of which influence the membrane association of the urea transporter, again through different mechanisms. Long-term regulation of urea transport is most closely associated with the environment that the kidney experiences. Low-protein diets may influence the amount of urea transporter available. Conditions of osmotic diuresis, where urea concentrations are low, will prompt an increase in urea transporter abundance. Although adrenal steroids affect urea transporter abundance, conflicting reports make conclusions tenuous. Urea transporters are upregulated when P2Y2 purinergic receptors are decreased, suggesting a role for these receptors in UT regulation. Hypercalcemia and hypokalemia both cause urine concentration deficiencies. Urea transporter abundances are reduced in aging animals and animals with angiotensin-converting enzyme deficiencies. This chapter will provide information about both rapid and long-term regulation of urea transporters and provide an introduction into the literature.

The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH(2)O. In contrast, urine osmolality was low (800 mosmol/kgH(2)O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.

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.

Regulation of water and urea transport in the inner medullary collecting duct is essential for urine concentration. Aquaporin (AQP)2 water channels and urea transporter (UT)-A1 are inserted into the apical membrane upon phosphorylation of the channels to allow the transcellular movement of water and urea. Since ANG II activates PKC in many cell types, we tested the hypothesis that ANG II-induced regulation of water and urea transport is mediated by PKC. Osmotic minipumps delivered ANG II to wild-type (WT) or PKC-α(-/-) mice for 7 days. Inner medullas were harvested, and protein abundance was determined by immunoblot. ANG II increased systolic blood pressure to a similar degree in WT and PKC-α(-/-) mice. ANG II had no effect on the urine output of WT mice but increased that of PKC-α(-/-) mice. In accordance with observed differences in urine output, AQP2 abundance was unchanged in ANG II-treated WT animals but was decreased in PKC-α(-/-) mice. No change in membrane accumulation was seen. Phosphorylation of the cAMP-induced transcription factor CREB was decreased in PKC-α(-/-) mice in response to ANG II with no change in overall CREB abundance. ANG II did not alter the abundance of UT-A1 protein in WT or PKC-α(-/-) mice. Phosphorylation and overall abundance of tonicity-responsive enhancer-binding protein, a transcription factor that regulates UT-A1, were also unaltered by ANG II in either group. We conclude that PKC-α protects against ANG II-induced decreases in urine concentrating ability by maintaining AQP2 levels through CREB phosphorylation.

Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats (n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS.

The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [(32)P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.

UT-A1, the urea transporter present in the apical membrane of the inner medullary collecting duct, is crucial to the kidney's ability to concentrate urine. Phosphorylation of UT-A1 on serines 486 and 499 is important for plasma membrane trafficking. The effect of calcineurin on dephosphorylation of UT-A1 was investigated. Inner medullary collecting ducts from Sprague-Dawley rats were metabolically labeled and treated with tacrolimus to inhibit calcineurin or calyculin to inhibit protein phosphatases 1 and 2A. UT-A1 was immunoprecipitated, electrophoresed, blotted, and total UT-A1 phosphorylation was assessed by autoradiography. Total UT-A1 was determined by Western blotting. A phospho-specific antibody to pser486-UT-A1 was used to determine whether serine 486 can be hyperphosphorylated by inhibiting phosphatases. Inhibition of calcineurin showed an increase in phosphorylation per unit protein at serine 486. In contrast, inhibition of phosphatases 1 and 2A resulted in an increase in UT-A1 phosphorylation but no increase in pser486-UT-A1. In vitro perfusion of inner medullary collecting ducts showed tacrolimus-stimulated urea permeability consistent with stimulated urea transport. The location of phosphorylated UT-A1 in rats treated acutely and chronically with tacrolimus was determined using immunohistochemistry. Inner medullary collecting ducts of the acutely treated rats showed increased apical membrane association of phosphorylated UT-A1 while chronic treatment reduced membrane association of phosphorylated UT-A1. We conclude that UT-A1 may be dephosphorylated by multiple phosphatases and that the PKA-phosphorylated serine 486 is dephosphorylated by calcineurin. This is the first documentation of the role of phosphatases and the specific site of phosphorylation of UT-A1, in response to tacrolimus.

The UT-A1 urea transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature N-acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation by tunicamycin or elimination of the glycosylation sites by mutation significantly reduced UT-A1 activity in oocytes. Taken together, our study reveals a new role of N-linked glycosylation in regulating UT-A1 activity by promoting UT-A1 trafficking into membrane lipid raft subdomains.

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/kgH(2)O 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.

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 NH(2) and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH(2) 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.

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.

Patients receiving lithium therapy, an effective treatment for bipolar disorder, often present with acquired nephrogenic diabetes insipidus. The nephrotoxic effects of lithium can be detected 3 wk after the start of treatment and many of these symptoms may disappear in a few weeks after lithium use is stopped. Most patients, however, still have a urine-concentrating defect years after ending treatment. This prompted an investigation of the transporters involved in the urine concentration mechanism, UT-A1, UT-A3, aquaporin-2 (AQP2), and NKCC2, after discontinuing lithium therapy. Sprague-Dawley rats fed a Li2CO3-supplemented diet produced large volumes of dilute urine after 14 days. After lithium treatment was discontinued, urine osmolality returned to normal within 14 days but urine volume and urine urea failed to reach basal levels. Western blot and immunohistochemical analyses revealed that both urea transporters UT-A1 and UT-A3 were reduced at 7 and 14 days of lithium treatment and both transporters recovered to basal levels 14 days after discontinuing lithium administration. Similar analyses demonstrated a decrease in AQP2 expression after 7 and 14 days of lithium therapy. AQP2 expression increased over the 7 and 14 days following the cessation of lithium but failed to recover to normal levels. NKCC2 expression was unaltered during the 14-day lithium regimen but did increase 14 days after the treatment was stopped. In summary, the rapid restoration of UT-A1 and UT-A3 as well as the increased expression of NKCC2 are critical components to the reestablishment of urine concentration after lithium treatment.

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.

The cell plasma membrane contains specialized microdomains called lipid rafts which contain high amounts of sphingolipids and cholesterol. Lipid rafts are involved in a number of membrane protein functions. The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we investigated the possible role of lipid rafts in UT-A1 membrane regulation. Using sucrose gradient cell fractionation, we demonstrated that UT-A1 is concentrated in the caveolae-rich fraction both in stably expressing UT-A1 HEK293 cells and in freshly isolated kidney IMCD suspensions. In these gradients, UT-A1 at the cell plasma membrane is codistributed with caveolin-1, a major component of caveolae. The colocalization of UT-A1 in lipid rafts/caveolae was further confirmed in isolated caveolae from UT-A1-HEK293 cells. The direct association of UT-A1 and caveolin-1 was identified by immunoprecipitation and GST pull-down assay. Examination of internalized UT-A1 in pEGFP-UT-A1 transfected HEK293 cells fluorescent overlap with labeled cholera toxin subunit B, a marker of the caveolae-mediated endocytosis pathway. Disruption of lipid rafts by methyl-beta-cyclodextrin or knocking down caveolin-1 by small-interference RNA resulted in UT-A1 cell membrane accumulation. Functionally, overexpression of caveolin-1 in oocytes decreased UT-A1 urea transport activity and UT-A1 cell surface expression. Our results indicate that lipid rafts/caveolae participate in UT-A1 membrane regulation and this effect is mediated via a direct interaction of caveolin-1 with UT-A1.

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.

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.

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 NaCl (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 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.

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.

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.

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

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.

Urea transport, mediated by the urea transporter A1 (UT-A1) and/or UT-A3, is important for the production of concentrated urine. Vasopressin rapidly increases urea transport in rat terminal inner medullary collecting ducts (IMCD). A previous study showed that one mechanism for rapid regulation of urea transport is a vasopressin-induced increase in UT-A1 phosphorylation. This study tests whether vasopressin or directly activating adenylyl cyclase with forskolin also increases UT-A1 accumulation in the plasma membrane of rat IMCD. Inner medullas were harvested from rats 45 min after injection with vasopressin or vehicle. UT-A1 abundance in the plasma membrane was significantly increased in the membrane fraction after differential centrifugation and in the biotinylated protein population. Vasopressin and forskolin each increased the amount of biotinylated UT-A1 in rat IMCD suspensions that were treated ex vivo. The observed changes in the plasma membrane are specific, as the amount of biotinylated UT-A1 but not the calcium-sensing receptor was increased by forskolin. Next, whether forskolin or the V(2)-selective agonist dDAVP would increase apical membrane expression of UT-A1 in MDCK cells that were stably transfected with UT-A1 (UT-A1-MDCK cells) was tested. Forskolin and dDAVP significantly increased UT-A1 abundance in the apical membrane in UT-A1-MDCK cells. It is concluded that vasopressin and forskolin increase UT-A1 accumulation in the plasma membrane in rat IMCD and in the apical plasma membrane of UT-A1-MDCK cells. These findings suggest that vasopressin regulates urea transport by increasing UT-A1 accumulation in the plasma membrane and/or UT-A1 phosphorylation.

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.

Mammalian urea transporters are facilitated membrane transport proteins belonging to two families, UT-A and UT-B. They are best known for their role of maintaining the renal inner medullary urinary concentrating gradient. Urea transporters have also been identified in tissues not typically associated with urea metabolism. The purpose of this study was to survey the major organs in rat to determine the distribution of UT-A and UT-B mRNA transcripts and protein forms and determine their cellular localization. Five kidney subregions and 17 extrarenal tissues were screened by Northern blot analysis using two UT-A and three UT-B probes and by Western blot analysis using polyclonal COOH-terminal UT-A and UT-B antibodies. Immunohistochemistry was performed on 16 extrarenal tissues using the same antibodies. In kidney, we detected mRNA transcripts and protein bands consistent with previously-identified UT-A and UT-B isoforms, as well as novel forms. We found that UT-A mRNA and protein are widely expressed in extrarenal tissues in various forms that are different from the known isoforms. We determined the cellular localization of UT-A and UT-B in these tissues. We found that both UT-A and UT-B are ubiquitously expressed as numerous tissue-specific mRNA transcripts and protein forms that are localized to cell membranes, cytoplasm, or nuclei.

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.

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kidd 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 approximately 50- and approximately 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 (14)C 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.

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.

Urea plays an important role in the urinary concentrating capacity. Renal inner medullary (IM) urea transporter expression was examined in rats with bilateral (BUO) or unilateral ureteral obstruction (UUO). BUO (24 h) was associated with markedly increased plasma urea (42.4 +/- 1.0 vs. 5.2 +/- 0.2 mmol/l) and a significant decrease in expression of UT-A1 (28 +/- 8% of sham levels), UT-A3 (45 +/- 11%), and UT-B (70 +/- 8%). Immunocytochemistry confirmed downregulation of UT-A1 and UT-A3 in IM collecting duct and UT-B in the descending vasa recta. Three days after release of BUO, UT-A1, UT-A3, and UT-B remained significantly downregulated (UT-A1: 37 +/- 6%; UT-A3: 25 +/- 6%; and UT-B: 10 +/- 5% of sham levels; P < 0.05) concurrent with a persistent polyuria and a marked reduction in solute-free water reabsorption (115 +/- 11 vs. 196 +/- 8 microl.min(-1).kg(-1), P < 0.05). Moreover, 14 days after release of BUO, total UT-A1, UT-A3, and UT-B remained significantly decreased compared with sham-operated controls and urine urea remained reduced (588 +/- 43 vs. 1,150 +/- 94 mmol/l). Consistent with increased levels of plasma urea 24 h after onset of UUO (7.4 +/- 0.3 vs. 4.8 +/- 0.3 mmol/l), the protein abundance of UT-A1, UT-A3, and UT-B in IM was markedly reduced in the obstructed kidney, which was confirmed by immunocytochemistry. In the nonobstructed kidney, the expression of urea transporters did not change. In conclusion, reduced expression of UT-A1, UT-A3, and UT-B levels in both BUO and UUO rats suggests that urea transporters play important roles in the impaired urinary concentrating capacity in response to urinary tract obstruction.

The UT-B urea transporter is the major urea transporter in red blood cells and kidney descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect. Whether deletion of UT-B altered the expression of other transporter proteins involved in urinary concentration was tested. Fluorescence-based real-time reverse transcription-PCR and Northern blot analysis showed upregulation of the UT-A2 urea transporter and the aquaporin 2 (AQP2) and AQP3 water channel transcripts but no change in other urea transporters or AQP. Western blot analysis showed that UT-A2 protein abundance in the outer medulla of UT-B null mice increased to 122 +/- 6% of wild-type control. AQP2 protein abundance increased to 177 +/- 32% and 127 +/- 7% in the outer and inner medulla, respectively, of UT-B null versus wild-type mice. The abundance of UT-A1, AQP1, renal outer medullary potassium channel, and NKCC2/BSC1 proteins were not significantly different between UT-B null and wild-type mice. The increases in AQP2 and AQP3 would reduce water loss and improve concentrating ability. The lack of UT-B does not result in a change in expression of urea transporters involved in urea reabsorption from the inner medullary collecting duct (UT-A1 and UT-A3). However, UT-B null mice have a selective increase in UT-A2 protein abundance. This may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.

Adrenalectomy in rats is associated with urinary concentrating and diluting defects. This study tested the effect of adrenal steroids on the UT-A1 urea transporter because it is involved in the urine-concentrating mechanism. Rats were adrenalectomized and given normal saline for 14 d, after which they received (1) vehicle, (2) aldosterone, or (3) spironolactone plus aldosterone. Adrenalectomy alone significantly increased UT-A1 protein in the inner medullary tip after 7 d, whereas aldosterone repletion reversed the effect. Spironolactone blocked the aldosterone-induced decrease in UT-A1, indicating that aldosterone was working via the mineralocorticoid receptor. For verifying that glucocorticoids downregulate UT-A1 protein through a different receptor, three groups of adrenalectomized rats were prepared: (1) vehicle, (2) adrenalectomy plus dexamethasone, and (3) adrenalectomy plus dexamethasone and spironolactone. Dexamethasone significantly reversed UT-A1 protein abundance increase in the inner medullary tip of adrenalectomized rats. When spironolactone was given with dexamethasone, it did not affect the dexamethasone-induced decrease in UT-A1. There was no significant change in serum vasopressin level, aquaporin 2, or Na(+)-K(+)-2Cl(-) co-transporter NKCC2/BSC1 protein abundances or UT-A1 mRNA abundance in any of the groups. In conclusion, either mineralocorticoids or glucocorticoids can downregulate UT-A1 protein. The decrease in UT-A1 does not require both steroid hormones, and each works through a different receptor.

We tested whether the abundance of transport proteins involved in the urinary concentrating mechanism was altered in rats with uncontrolled diabetes mellitus (DM). Rats were injected with streptozotocin and killed 5, 10, 14, or 20 days later. Blood glucose in DM rats was 300-450 mg/dl (control: 70-130 mg/dl). Urine volume increased in DM rats from 41 +/- 7 ml/100 g body wt (BW) at 5 days to 69 +/- 3 ml/100 g BW at 20 days (control: 9 +/- 1). Urine osmolality of DM rats decreased at 5 days DM and remained low at 20 days. UT-A1 urea transporter protein in the inner medullary (IM) tip was 55% of control in 5-day DM rats but increased to 170, 220, and 280% at 10, 14, and 20 days DM, respectively, due to an increase in the 117-kDa glycoprotein form. UT-A1 in the IM base was increased to 325% of control at 5 days DM with no further increase at 20 days. Aquaporin-2 (AQP2) increased to 290% in the IM base at 5 days DM and 150% in the IM tip at 10 days; both showed no further increase at 20 days. NKCC2/BSC1 increased to 240% in outer medulla at 20 days DM, but not at 5 or 10 days. UT-B and ROMK were unchanged at any time point. The increases in UT-A1, AQP2, and NKCC2/BSC1 proteins during uncontrolled DM would tend to limit the loss of fluid and solute during uncontrolled diabetes.

Our laboratory previously reported that uremic levels of urea inhibit L-arginine (L-Arg) transport into endothelial cells. The present study further investigated this effect. We measured L-Arg transport in cultured bovine aortic endothelial cells with normal or high urea (25 mM). The urea transport inhibitor phloretin abolished the inhibitory effect of urea on L-Arg transport, suggesting a role for urea transporters (UTs). We screened bovine aortic endothelial cells and several other endothelial cell types for the presence of UTs by using Western blot analysis. UT-B was present in all endothelial cells, irrespective of species or location of derivation, whereas UT-A distribution was variable and sparse. UT-B was also abundant in rat aorta, mesenteric blood vessels, and spinotrapezius muscle, whereas UT-A distribution was, again, variable and sparse. Chronic elevation of urea had variable, inconsistent effects on UT abundance. This study showed that urea must enter endothelial cells, probably by UT-B, to inhibit L-Arg transport. In view of the wide distribution of UT-B in rat vasculature, elevated blood urea nitrogen may lead to endothelial L-Arg deficiency in vivo.

Liver expresses a 49-kD UT-A protein whose abundance is increased by uremia. Chronic renal failure causes acidosis; therefore, the role of acidosis in increasing UT-A abundance was tested. Rats underwent 5/6 nephrectomy, and half were given bicarbonate mixed in their food. Bicarbonate administration significantly increased blood pH. Compared with sham-operated rats, UT-A protein abundance was significantly increased by 50% in livers from uremic, acidotic rats; bicarbonate administration prevented the increase in UT-A protein. To determine whether acidosis alone would increase UT-A protein in liver, rats were made acidotic, but not uremic, by feeding them HCl. HCl-feeding significantly lowered blood pH, increased urea excretion, and increased the abundance of the 49-kD liver UT-A protein by 36% compared with pair-fed nonacidotic rats. HCl-feeding significantly increased the abundance of the 117-kD UT-A1 protein in kidney inner medulla but did not change aquaporin-2 protein. Next, rats were fed urea to determine whether elevated blood urea would increase UT-A protein. However, urea feeding had no effect on UT-A in liver or kidney inner medulla. It was, therefore, concluded that acidosis, either directly or through a change in ammonium concentration, rather than other dietary components, stimulates the upregulation of UT-A protein in liver and kidney inner medulla.

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.