Molecular approaches to urea transporters

Urea plays a critical role in the urine-concentrating mechanism in the inner medulla. Physiologic data provided evidence that urea transport in red blood cells and kidney inner medulla was mediated by specific urea transporter proteins. Molecular approaches during the past decade resulted in the cloning of two gene families for facilitated urea transporters, UT-A and UT-B, encoding several urea transporter cDNA isoforms in humans, rodents, and several nonmammalian species. Polyclonal antibodies have been generated to the cloned urea transporter proteins, and the use of these antibodies in integrative animal studies has resulted in several novel findings, including: (1) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; (2) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (3) UT-A protein abundance is upregulated in uremia in both liver and heart; and (4) UT-B is expressed in many nonrenal tissues and endothelial cells. This review will summarize the knowledge gained from using molecular approaches to perform integrative studies into urea transporter protein regulation, both in normal animals and in animal models of human diseases, including studies of uremic rats in which urea transporter protein is upregulated in liver and heart.     

Vasopressin increases plasma membrane accumulation of urea transporter UT-A1 in rat inner medullary collecting ducts

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.     

Regulation of renal urea transporters

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.     

Downregulation of UT-A1/UT-A3 is associated with urinary concentrating defect in glucocorticoid-excess state

Excessive glucocorticoid hormone, as occurs with Cushing syndrome, is known to be associated with altered body water homeostasis, but the molecular mechanisms are unknown. In this study, rats treated with daily dexamethasone (Dex) for 14 d provided a model of Cushing syndrome. Compared with control rats, Dex-treated rats demonstrated increased mean arterial pressure, urine flow rate, and urinary excretion of both sodium and urea. Dex-treated rats had increased abundance of aquaporin 1 (AQP1), AQP3, and Na-K-2Cl co-transporter proteins and a marked reduction of the urea transporters UT-A1 and UT-A3. In response to an acute water load, Dex-treated rats increased water excretion more than control rats, but both groups exhibited similar AQP2 expression. In response to fluid deprivation, Dex-treated rats demonstrated an impaired urinary concentrating capacity: Urine flow rate was higher and urine osmolality was lower than control rats despite an increase in AQP1, AQP3, and Na-K-2Cl co-transporter expression. AQP2 expression was similar between the two groups, but UT-A1 and UT-A3 were decreased and urinary urea excretion was increased in Dex-treated rats. Because Dex-treated rats ingested less food and water compared with controls, paired food and water studies were performed; these substantiated the previous results. In summary, the alterations in body water observed with glucocorticoid excess may be a result, in part, of impaired urinary concentrating capacity; downregulation of UT-A1 and UT-A3 and increased urea excretion may contribute to this impairment.     

Massive reduction of urea transporters in remnant kidney and brain of uremic rats

BACKGROUND: The facilitated urea transporters (UT), UT-A1, UT-A2, and UT-B1, are involved in intrarenal recycling of urea, an essential feature of the urinary concentrating mechanism, which is impaired in chronic renal failure (CRF). In this study, the expression of these UTs was examined in experimentally induced CRF. METHODS: The abundance of mRNA was measured by Northern analysis and that of corresponding proteins by Western blotting in rats one and five weeks after 5/6 nephrectomy (Nx). RESULTS: At five weeks, urine output was enhanced threefold with a concomitant decrease in urine osmolality. The marked rise in plasma urea concentration and fall in urinary urea concentration resulted in a 30-fold decrease in the urine/plasma (U/P) urea concentration ratio, while the U/P osmoles ratio fell only fourfold. A dramatic decrease in mRNA abundance for the three UTs was observed, bringing their level at five weeks to 1/10th or less of control values. Immunoblotting showed complete disappearance of the 97 and 117 kD bands of UT-A1, and considerable reduction of UT-A2 and UT-B1 in the renal medulla. Similar, but less intense, changes were observed at one-week post-Nx. In addition to the kidney, UT-B1 is also normally expressed in brain and testis. In the brain, its mRNA expression remained normal one-week post-Nx, but decreased to about 30% of normal at five-weeks post-Nx, whereas no change was seen in testis. CONCLUSIONS: (1) The decline in urinary concentrating ability seen in CRF is largely due to a major reduction of UTs involved in the process of urea concentration in the urine, while factors enabling the concentration of other solutes are less intensely affected. (2) The marked reduction of brain UT expression in CRF may be responsible for brain edema of dialysis disequilibrium syndrome observed in some patients after fast dialysis.     

Urea and renal function in the 21st century: insights from knockout mice

Since the turn of the 21st century, gene knockout mice have been created for all major urea transporters that are expressed in the kidney: the collecting duct urea transporters UT-A1 and UT-A3, the descending thin limb isoform UT-A2, and the descending vasa recta isoform UT-B. This article discusses the new insights that the results from studies in these mice have produced in the understanding of the role of urea in the urinary concentrating mechanism and kidney function. Following is a summary of the major findings: (1) Urea accumulation in the inner medullary interstitium depends on rapid transport of urea from the inner medullary collecting duct (IMCD) lumen via UT-A1 and/or UT-A3; (2) as proposed by Robert Berliner and colleagues in the 1950s, the role of IMCD urea transporters in water conservation is to prevent a urea-induced osmotic diuresis; (3) the absence of IMCD urea transport does not prevent the concentration of NaCl in the inner medulla, contrary to what would be predicted from the passive countercurrent multiplier mechanism in the form proposed by Kokko and Rector and Stephenson; (4) deletion of UT-B (vasa recta isoform) has a much greater effect on urinary concentration than deletion of UT-A2 (descending limb isoform), suggesting that the recycling of urea between the vasa recta and the renal tubules quantitatively is less important than classic countercurrent exchange; and (5) urea reabsorption from the IMCD and the process of urea recycling are not important elements of the mechanism of protein-induced increases in GFR. In addition, the clinical relevance of these studies is discussed, and it is suggested that inhibitors that specifically target collecting duct urea transporters have the potential for clinical use as potassium-sparing diuretics that function by creation of urea-dependent osmotic diuresis.     

Molecular mechanisms of impaired urinary concentrating ability in glucocorticoid-deficient rats

The purpose of this study was to examine urinary concentrating ability and protein expression of renal aquaporins and ion transporters in glucocorticoid-deficient (GD) rats in response to water deprivation as compared with control rats. Rats underwent bilateral adrenalectomies, followed only by aldosterone replacement (GD) or both aldosterone and dexamethasone replacement (control). As compared with control rats, the GD rats demonstrated a decrease in cardiac output and mean arterial pressure. In response to 36-h water deprivation, GD rats demonstrated significantly greater urine flow rate and decreased urine osmolality as compared with control rats at comparable serum osmolality and plasma vasopressin concentrations. The initiator of the countercurrent concentrating mechanism, the sodium-potassium-2 chloride co-transporter, was significantly decreased, as was the medullary osmolality in the GD rats versus control rats. There was also a decrease in inner medulla aquaporin-2 (AQP2) and urea transporter A1 (UT-A1) in GD rats as compared with control rats. There was a decrease in outer medulla Gsalpha protein, an important factor in vasopressin-mediated regulation of AQP2. Immunohistochemistry studies confirmed the decreased expression of AQP2 and UT-A1 in kidneys of GD rats as compared with control. In summary, impairment in the urinary concentrating mechanism was documented in GD rats in association with impaired countercurrent multiplication, diminished osmotic equilibration via AQP2, and diminished urea equilibration via UT-A1. These events occurred primarily in the relatively oxygen-deficient medulla and may have been initiated, at least in part, by the decrease in mean arterial pressure and thus renal perfusion pressure in this area of the kidney.     

Urea transporters and renal function: lessons from knockout mice

PURPOSE OF REVIEW: Gene knockout mice have been created for the collecting duct urea transporters UT-A1 and UT-A3, the descending thin-limb urea transporter UT-A2 and the descending vasa recta isoform, UT-B. In this brief review, the new insights in our understanding of the role of urea in the urinary concentrating mechanism and kidney function resulting from studies in these mice are discussed. RECENT FINDINGS: The major findings in studies on urea transporter knockout mice are as follows: rapid transport of urea from the inner medulla collecting duct lumen via UT-A1 or UT-A3 is essential for urea accumulation in the inner medullary interstitium; inner medulla collecting duct urea transporters are essential in water conservation by preventing urea-induced osmotic diuresis; an absence of inner medulla collecting duct urea transport does not prevent the concentration of sodium chloride in the inner medulla interstitium; deletion of the vasa recta isoform UT-B has a much greater effect on urinary concentration than deleting the descending limb isoform UT-A2. SUMMARY: Multiple urea transport mechanisms within the kidney are essential for producing maximally concentrated urine.     

Renal phenotype of UT-A urea transporter knockout mice

The urea transporters UT-A1 and UT-A3 mediate rapid transepithelial urea transport across the inner medullary collecting duct (IMCD). In a previous study, using a new mouse model in which both UT-A1 and UT-A3 were genetically deleted from the IMCD (UT-A1/3(-/-) mice), we investigated the role of these transporters in the function of the renal inner medulla. Here the authors report a new series of studies investigating more generally the renal phenotype of UT-A1/3(-/-) mice. Pathologic screening of 33 tissues revealed abnormalities in both the testis (increased size) and kidney (decreased size and vascular congestion) of UT-A1/3(-/-) mice. Total urinary nitrate and nitrite (NOx) excretion rates in UT-A1/3(-/-) mice were more than double those in wild-type mice. Total renal blood flow was not different between UT-A1/3(-/-) and wild-type mice but underwent a greater percentage decrease in response to NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME) infusion. Whole kidney GFR (FITC-inulin clearance) was not different in UT-A1/3(-/-) mice compared with controls and underwent a similar increase in response to a greater dietary protein intake. Fractional urea excretion was markedly elevated in UT-A1/3(-/-) mice on a 40% protein diet, reaching 102.4 +/- 8.8% of the filtered load, suggesting that there may be active urea secretion somewhere along the renal tubule. Although there was a marked urinary concentrating defect in UT-A1/3(-/-) mice, there was no decrease in aquaporin 2 or aquaporin 3 expression. Furthermore, although urea accumulation in the inner medulla was markedly attenuated, there was no decrease in sodium ion concentration in tissue from outer medulla or two levels of the inner medulla. These results support our conclusion that the urinary concentrating defect in UT-A1/3(-/-) mice is caused by a failure of urea transport from the IMCD lumen to the inner medullary interstitium, resulting in osmotic diuresis.     

Molecular basis for the dialysis disequilibrium syndrome: altered aquaporin and urea transporter expression in the brain.

BACKGROUND: Cerebral disorders caused by brain oedema characterize the dialysis disequilibrium syndrome, a complication of rapid haemodialysis. Brain oedema is presumably caused by the 'reverse urea effect', i.e. the significant urea gradient between blood and brain after dialysis, with, as a result, an inflow of water into the brain. To assess the molecular basis of this effect, we examined the expression of urea transporter UT-B1 and aquaporin (AQP) 4 and AQP9 in the brain of uraemic rats. METHODS: Brain, kidneys and one testis were collected from four sham-operated (control) and four uraemic rats, 10 weeks after 5/6 nephrectomy (Nx). Protein abundance was measured by semi-quantitave immunoblotting using affinity-purified rabbit anti-rat antibodies applied on tissue crude homogenates. RESULTS: The results are expressed as means+/-SE of band density (arbitrary units). In Nx compared with control rats, the brain expression of UT-B1 was reduced by half (32+/-3 vs 62+/-8, P<0.01) whereas that of AQ4 was doubled (251+/-13 vs 135+/-5, P<0.001), and that of AQP9 increased by 65% (253+/-22 vs 154+/-10, P<0.01). UT-B1 expression was also lowered by Nx in kidney medulla (45+/-21 vs 141+/-4, P<0.01) but was unchanged in testis. CONCLUSIONS: The conjunction of a reduced expression of UT-B and an increased expression of AQPs in brain cells may bring a new clue to understanding the DDS mechanism. Because of low UT-B abundance, urea exit from astrocytes is most probably delayed during rapid removal of extracellular urea through fast dialysis. This creates an osmotic driving force that promotes water entry into the cells (favoured by abundant AQPs) and subsequent brain swelling.     

Urinary concentrating defect in hypothyroid rats: role of sodium,potassium, 2-chloride co-transporter,and aquaporins

Hypothyroidism is associated with impaired urinary concentrating ability in humans and animals. The purpose of this study was to examine protein expression of renal sodium chloride and urea transporters and aquaporins in hypothyroid rats (HT) with diminished urinary concentration as compared with euthyroid controls (CTL) and hypothyroid rats replaced with L-thyroxine (HT+T). Hypothyroidism was induced by aminotriazole administration. Body weight, water intake, urine output, solute and urea excretion, serum and urine osmolality, serum creatinine, 24-h creatinine clearance, and fractional excretion of sodium were comparable among the three groups. However, with 36 h of water deprivation, HT rats demonstrated significantly greater urine flow rates and decreased urine and medullary osmolality as compared with CTL and HT+T rats at comparable plasma vasopressin concentrations. Western blot analyses revealed decreased renal protein abundance of transporters, including Na-K-2Cl, Na-K-ATPase, and NHE3, in HT rats as compared with CTL and HT+T rats. Protein abundance of renal AQP1 and urea transporters UTA(1) and UTA(2) did not differ significantly among study groups. There was however a significant decrease in protein abundance of AQP2, AQP3, and AQP4 in HT rats as compared with CTL and HT+T rats. These findings demonstrate a decrease in the medullary osmotic gradient secondary to impaired countercurrent multiplication and downregulation of aquaporins 2, 3, and 4 as contributors to the urinary concentrating defect in the hypothyroid rat.     

Expression of salt and urea transporters in rat kidney during cisplatin-induced polyuria.

BACKGROUND: Cisplatin (CP) induced polyuria in rats is associated with a reduction in medullary hypertonicity, normally generated by the thick ascending limb (TAL) salt transporters, and the collecting duct urea transporters (UT). To investigate the molecular basis of this abnormality, we determined the protein abundance of major salt and UT isoforms in rat kidney during CP-induced polyuria. METHODS: Male Sprague-Dawley rats received either a single injection of CP (5 mg/kg, N = 6) or saline (N = 6) intraperitoneally five days before sacrifice. Urine, blood, and kidneys were collected and analyzed. RESULTS: CP-treated rats developed polyuric acute renal failure as assessed by increased blood urea nitrogen (BUN), urine volume and decreased urine osmolality. Western analysis of kidney homogenates revealed a marked reduction in band density of the bumetanide-sensitive Na-K-2Cl cotransporter in cortex (60% of control values, P < 0.05), but not in outer medulla (OM) (106% of control values). There were no differences in band densities for the renal outer medullary potassium channel (ROMK), the type III Na-H exchanger (NHE3), the alpha-subunit of Na,K-ATPase in the OM; or for UT-A1, UT-A2 or UT-A4 in outer or inner medulla. However, the band pattern of UT-A2 and UT-A4 proteins in the OM of CP-treated rats was different from the control rats, suggesting a qualitative modification of these proteins. CONCLUSIONS: Changes in the abundance of outer or inner medullary salt or urea transporters are unlikely to play a role in the CP-induced reduction in medullary hypertonicity. However, qualitative changes in UT proteins may affect their functionality and thus may have a role.     

Triazolothienopyrimidine inhibitors of urea transporter UT-B reduce urine concentration

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.     

Aldosterone decreases UT-A1 urea transporter expression via the mineralocorticoid receptor

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.     

Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4

Urea transport in the kidney is important for the production of concentrated urine and is mediated by a family of transporter proteins, identified from erythropoietic tissue (UT-B) and from kidney (UT-A). Two isoforms of the renal urea transporter (UT-A) have been cloned so far: UT-A1 and UT-A2. We used rapid amplification of cDNA ends to clone two new isoforms of the rat UT-A transporter: UT-A3 and UT-A4. UT-A3 and UT-A4 are 87% homologous. The UT-A3 cDNA encodes a peptide of 460 amino acids, which corresponds to the amino-terminal half of the UT-A1 peptide and is 62% identical to UT-A2. The UT-A4 cDNA encodes a peptide of 466 amino acids, which is 84% identical to UT-A2. Transient transfection of HEK-293 cells with the UT-A3 or UT-A4 cDNA results in phloretin-inhibitable urea uptake, which is increased by forskolin. Thus, both new isoforms encode functional urea transporters that may be vasopressin-regulated. UT-A3 and UT-A4 mRNA are expressed in the renal outer and inner medulla but not in the cortex; unidentified UT-A isoforms similar to UT-A3 may also be expressed in the testis. It is concluded that there are at least four different rat UT-A urea transporters.     

Acidosis mediates the upregulation of UT-A protein in livers from uremic rats

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.     

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

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.Urea plays a crucial role in the urinary concentrating mechanism, and therefore, in the regulation of water balance. Urea''s importance to the generation of a concentrated urine has been appreciated since at least 1934.^1,2 Several studies have shown that maximal urine concentrating ability is decreased in protein-deprived mammals and is restored by urea infusion.³ More recently, a UT-A1/UT-A3 knock-out mouse,^4,5 a UT-A2 knock-out mouse,⁶ and a UT-B knock-out mouse^7–9 were each shown to have urine concentrating defects. Thus, any hypothesis regarding the mechanism by which the kidney concentrates urine needs to include some effect derived from urea.The UT-A1 urea transporter is expressed in the terminal inner medullary collecting duct (IMCD).¹⁰ Vasopressin stimulates urea transport across perfused rat terminal IMCDs by increasing UT-A1 phosphorylation and apical plasma membrane accumulation.^11–15 Vasopressin acts by binding to V₂ receptors in the basolateral plasma membrane, stimulating adenylyl cyclase, increasing cAMP production, and increasing urea transport.^11,16–18 Forskolin, which directly activates adenylyl cyclase,¹⁹ also increases urea transport in perfused rat terminal IMCDs.²⁰cAMP is traditionally thought to act through protein kinase A (PKA). However, when we stimulate the PKA activity by increasing cAMP with forskolin in MDCK cells that are stably transfected with UT-A1 (UT-A1-MDCK cells), only 50% of the forskolin-stimulated urea flux is inhibited by H-89, a PKA inhibitor.²¹ Vasopressin and forskolin work in a similar manner to increase the cAMP levels, so this partial inhibition by H-89 suggests that vasopressin may signal through two cAMP-dependent pathways: one involving PKA and one that is independent of PKA. Because the UT-A1-MDCK cells reproduce many of the properties of native rat IMCDs,^13,21,22 these findings raise the possibility that vasopressin may signal through a second cAMP-dependent, but non–PKA-dependent, pathway in rat IMCDs.In addition to PKA, cAMP can activate Epac (exchange protein activated by cAMP), which signals by activating Rap1, a Ras-related small molecular weight G protein, which in turn signals through mitogen-activated protein kinase kinase (MEK) and extracellular signal-related kinase (ERK)^23,24 (Figure 1). There are two closely related exchange proteins activated by cAMP (Epac) proteins, Epac1 and Epac2, and both have been detected in rat IMCDs, although one or the other predominates in different studies.^25–28 The purpose of this study was to determine whether activation of the Epac pathway resulted in a functional change in urea transport in perfused rat terminal IMCDs.Open in a separate windowFigure 1.Vasopressin signaling diagram.     

UT-A urea transporter protein expressed in liver: upregulation by uremia.

In perfused rat liver, there is phloretin-inhibitable urea efflux, but whether it is mediated by the kidney UT-A urea transporter family is unknown. To determine whether cultured HepG2 cells transport urea, thiourea influx was measured. HepG2 cells had a thiourea influx rate of 1739 +/- 156 nmol/g protein per min; influx was inhibited 46% by phloretin and 32% by thionicotinamide. Western analysis of HepG2 cell lysate using an antibody to UT-A1, UT-A2, and UT-A4 revealed two protein bands: 49 and 36 kD. The same bands were detected in cultured rat hepatocytes, freshly isolated rat hepatocytes, and in liver from rat, mouse, and chimpanzee. Both bands were present when analyzed by native gel electrophoresis, and deglycosylation of rat liver lysate had no effect on either band. Differential centrifugation of rat liver lysate showed that the 49-kD protein is in the membrane fraction and the 36-kD protein is in the cytoplasm. To determine whether the abundance of these UT-A proteins varies in vivo, rats were made uremic by 5/6 nephrectomy. The 49-kD protein was significantly increased 5.5-fold in livers from uremic rats compared to pair-fed control rats. It is concluded that phloretin-inhibitable urea flux in liver may occur via a 49-kD protein that is specifically detected by a UT-A antibody. Uremia increases the abundance of this 49-kD UT-A protein in rat liver in vivo.