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Urea transporters are membrane proteins that selectively allow urea molecules to pass through. It is not clear how these transporters allow rapid conduction of urea, a polar molecule, in spite of the presence of a hydrophobic constriction lined by aromatic rings. The current study elucidates the mechanism that is responsible for this rapid conduction by performing free energy calculations on the transporter dvUT with a cumulative sampling time of about 1.3 μs. A parallel arrangement of aromatic rings in the pore enables stacking of urea with these rings, which, in turn, lowers the energy barrier for urea transport. Such interaction of the rings with urea is proposed to be a conserved mechanism across all urea-conducting proteins. The free energy landscape for the permeation of multiple urea molecules reveals an interplay between interurea interaction and the solvation state of the urea molecules. This is for the first time that multiple molecule permeation through any small molecule transporter has been modeled.

The symbiotic relationship between humans and their intestinal microbiomeis supported by urea nitrogen salvaging. Previous studies have shown thatcolonic UT-B urea transporters play a significant role in this important physi-ological process. This current study investigated UT-A and UT-B urea trans-porter expression along the human gastrointestinal tract. Initial end-pointPCR experiments determined that UT-A RNA was predominantly expressedin the small intestine, while UT-B RNA was expressed in stomach, small intes-tine, and colon. Using western blotting experiments, a strong 40–60 kDa UT-B signal was found to be abundant in both ileum and colon. Importantly, thissignal was deglycosylated by PNGaseF enzyme treatment to a core protein of30 kDa in both tissues. Further immunolocalization studies revealed UT-Btransporter proteins were present at the apical membrane of the villi in theileum, but predominantly at the basolateral membrane of the colonic surfaceepithelial cells. Finally, a blind scoring immunolocalization study suggestedthat there was no significant difference in UT-B abundance throughout thecolon (NS, ANOVA,N=5–21). In conclusion, this current study suggestedUT-B to be the main human intestinal urea transporter. Intriguingly, thesedata suggested that the same UT-B isoform was present in all intestinalepithelial cells, but that the precise cellular location varied. Science Foundation Ireland

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics (‘urearetics’) with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10–20 μM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2–3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 hours, and a urine concentration of 20–40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Urea transporters (UT) play an important role in the urine concentration mechanism by mediating intrarenal urea recycling, suggesting that UT inhibitors could have therapeutic use as a novel class of diuretic. Recently, we found a thienoquinolin UT inhibitor, PU-14, that exhibited diuretic activity. The purpose of this study was to identify more potent UT inhibitors that strongly inhibit UT-A isoforms in the inner medullary collecting duct (IMCD). Efficient thienoquinolin UT inhibitors were identified by structure-activity relationship analysis. Urea transport inhibition activity was assayed in perfused rat terminal IMCDs. Diuretic activity of the compound was determined in rats and mice using metabolic cages. The results show that the compound PU-48 exhibited potent UT-A inhibition activity. The inhibition was 69.5% with an IC50of 0.32 μM. PU-48 significantly inhibited urea transport in perfused rat terminal IMCDs. PU-48 caused significant diuresis in UT-B null mice, which indicates that UT-A is the target of PU-48. The diuresis caused by PU-48 did not change blood Na+, K+, or Cl−levels or nonurea solute excretion in rats and mice. No toxicity was detected in cells or animals treated with PU-48. The results indicate that thienoquinolin UT inhibitors induce a diuresis by inhibiting UT-A in the IMCD. This suggests that they may have the potential to be developed as a novel class of diuretics with fewer side effects than classical diuretics.

The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance phase could also be an adaptive feature to prepare for efficient urea excretion when water becomes available.

In mammals, urea is the main nitrogenous breakdown product of protein catabolism and is produced in the liver. In certain tissues, the movement of urea across cell membranes is specifically mediated by a group of proteins known as the SLC14A family of facilitative urea transporters. These proteins are derived from two distinct genes, UT-A (SLC14A2) and UT-B (SLC14A1). Facilitative urea transporters play an important role in two major physiological processes - urinary concentration and urea nitrogen salvaging. Although UT-A and UT-B transporters both have a similar basic structure and mediate the transport of urea in a facilitative manner, there are a number of significant differences between them. UT-A transporters are mainly found in the kidney, are highly specific for urea, have relatively lower transport rates and are highly regulated at both gene expression and cellular localization levels. In contrast, UT-B transporters are more widespread in their tissue location, transport both urea and water, have a relatively high transport rate, are inhibited by mercurial compounds and currently appear to be less acutely regulated. This review details the fundamental research that has so far been performed to investigate the function and physiological significance of these two types of urea transporters.

Ruminants are known to be able to very effectively recycle urinary urea and reuse it as a source of N for ruminal microbes. It is presumed that urea recycling is accomplished by specialized urea transporters (UT) which are localized in the kidney. This could be especially important in times of increased N requirement, such as during growth or during reduced dietary N intake. The aim of our study was to characterize and to localize UT in the goat (capra hircus) kidney and to investigate its response to reduced dietary N intake in growing goats. Therefore, 12 growing, male goats were fed either a diet containing high (17% CP in complete diet) or low (9% CP in complete diet) N content for 6 wk. After harvesting, blood and kidney samples were taken and analyzed. The mRNA of the different UT isoforms, UT-A1, UT-A2 and UT-B, were detected semiquantitatively in renal tissue by Northern blot analysis. For UT-A2 and UT-B, no statistically significant effect of dietary N restriction on renal mRNA expression could be detected (UT-A2: P = 0.26, UT-B: P = 0.07). However, renal mRNA abundance of UT-A1 significantly increased in the kidney of low-N-fed goats (P = 0.01). Furthermore, protein amounts of UT-B were verified by western blotting; and the localization of UT-A2 and UT-B protein was demonstrated by immunohistochemistry. No significant differences in protein amounts of UT-B could be observed comparing the 2 feeding groups (P = 0.78). The UT-B was localized in renal medulla and papilla, whereas UT-A2 was only found in renal medulla. In addition, comparison of UT-A and UT-BAA sequences of monogastric animals and ruminants showed a high degree of homology, indicating a similar function of the transporters among these species. In summary, we conclude that in ruminants, urea reabsorption in the kidney is most likely increased in response to a low-N diet via an upregulation of UT-A1 mRNA expression. Hypothetically, the reabsorbed urea can then be returned to the rumen via the bloodstream and thus be reused as a source of N for protein synthesis of ruminal microbial community.

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 decease 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 μl·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.

Acute renal failure (ARF) is a frequent complication of sepsis and has a high mortality. Sepsis-induced ARF is known to be associated with significant impairment of tubular capacity. However, the pathogenesis of endotoxemic tubular dysfunction with failure of urine concentration is poorly understood. Urea plays an important role in the urinary concentrating mechanism and expression of the urea transporters UT-A1, UT-A2, UT-A3, UT-A4, and UT-B is essential for tubular urea reabsorption. The present study attempts to assess the regulation of renal urea transporters during severe inflammation in vivo. Lipopolysaccharide-(LPS)-injected mice presented with reduced glomerular filtration rate, fractional urea excretion, and inner medulla osmolality associated with a marked decrease in expression of all renal urea transporters. Similar alterations were observed after application of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, or IL-6. LPS-induced downregulation of urea transporters was not affected in knockout mice with deficient TNF-alpha, IL-receptor-1, IFN-gamma, or IL-6. Glucocorticoid treatment inhibited LPS-induced increases of tissue TNF-alpha, IL-1beta, IFN-gamma, or IL-6 concentration, diminished LPS-induced renal dysfunction, and attenuated the downregulation of renal urea transporters. Renal ischemia induced by renal artery clipping did not influence the expression of urea transporters. Our data demonstrate that renal urea transporters are downregulated by severe inflammation, which likely accounts for tubular dysfunction. Furthermore, they suggest that the downregulation of renal urea transporters during LPS-induced ARF is mediated by proinflammatory cytokines and is independent from renal ischemia because of sepsis-induced hypotension.

Urea movement across plasma membranes is modulated by specialized urea transporter proteins. These proteins are proposed to play key roles in the urinary concentrating mechanism and fluid homeostasis. To date, two urea-transporter genes have been cloned; UT-A (Slc14a2), encoding at least five proteins and UT-B (Slc14a1) encoding a single protein isoform. Recently we engineered mice that lack the inner medullary collecting duct (IMCD) urea transporters, UT-A1 and UT-A3 (UT-A1/3 −/− mice). This article includes 1) a historical review of the role of renal urea transporters in renal function; 2) a review of our studies utilizing the UT-A1/3 −/− mice; 3) description of an additional line of transgenic mice in which beta-galactosidase expression is driven by the alpha-promoter of the UT-A gene, which is allowing better physiological definition of control mechanisms for UT-A expression; and 4) a discussion of the implications of the studies in transgenic mice for the teaching of kidney physiology.

Urea transporters (UTs) encoded by the Slc14a1 (UT-B) and Slc14a2 (UT-A) genes mediate urea flux across cellular membranes. Considerable research has accrued detailing the function and distribution of members of both subfamilies. Much research effort has focused on the kidney, where UTs are highly expressed and function to promote urine concentration. Interestingly, UTs are also expressed in several other tissues that are historically not primarily associated with urea metabolism. In this review, I describe the phenotypes of UT knockout and transgenic mice and highlight the major advances made possible by use of these animal models. Where pertinent, I contrast these findings with known human phenotypes associated with UT mutations.

Urea transport in the kidney is important for the production of concentrated urine. This process is mediated by urea transporters (UTs) encoded by two genes, UT-A (Slc14a2) and UT-B (Slc14a1). Our previous study demonstrated that cetaceans produce highly concentrated urine than terrestrial mammals, and that baleen whales showed higher concentrations of urinary urea than sperm whales. Therefore, we hypothesized that cetaceans have unique actions of UTs to maintain fluid homeostasis in marine habitat. Kidney samples of common minke (Balaenoptera acutorostrata), sei (B. borealis), Bryde's (B. brydei) and sperm whales (Physeter macrocephalus) were obtained to determine the nucleotide sequences of mRNAs encoding UT. The sequences of 2.5-kb cDNAs encode 397-amino acid proteins, which are 90-94% identical to the mammalian UT-A2s. Two putative glycosylation sites are conserved between the whales and the terrestrial mammals, whereas consensus sites for protein kinases are not completely conserved; only a single protein kinase A consensus site was identified in the whale UT-A2s. Two protein kinase C consensus sites are present in the baleen whale UT-A2s, however, a single protein kinase C consensus site was identified in the sperm whale UT-A2. These different phosphorylation sites of whale UT-A2s may result in the high concentrations of urinary urea in whales, by reflecting their urea permeability.

One of the two phloretin-sensitive, facilitated urea transporters identified from the kidneys of the myliobatiform, euryhaline elasmobranch, Dasyatis sabina, a 379 amino acid protein ([D. sabina]strUT-2), was very similar to the 380 amino acid isoform (shUT) present in the kidney of the squaliform, dogfish shark, Squalus acanthias (a species that can be considered marginally euryhaline since it utilizes upper estuarine, as well as ocean habitats). To test the proposal that this isoform is a conserved urea transporter (UT) expressed in the kidneys of diverse elasmobranchs, UTs were cloned from the kidneys of a rajiform elasmobranch, the stenohaline skate, Leucoraja ocellata and another dasyatid stingray, the marginally euryhaline, Dasyatis say. Utilizing 5′/3′ RACE, a 2,060 nt cDNA that encoded a phloretin-sensitive, 378 amino acid skate urea transporter ([L. ocellata]skUT-2) and a 1,683 nt cDNA that encoded a stingray 379 amino acid UT ([D. say]strUT-2) were obtained. These deduced UTs have a very high sequence identity with the known elasmobranch Uts. [L. ocellata]skUT-2 was 86% identical to [D. sabina]strUT-2 and 84% identical to [S. acanthias]shUT. [D. say]strUT-2 was 97% identical to the [D. sabina]strUT-2. These findings support the hypothesis that a conserved UT isoform is present in the kidneys of marine elasmobranchs, and is an important pathway for facilitated urea transport in the kidneys of marine elasmobranchs.

Purpose of review Urea is transported across the kidney inner medullary collecting duct by urea-transporter proteins. Two urea-transporter genes have been cloned from humans and rodents: the UT-A (Slc14A2) gene encodes five protein and eight cDNA isoforms; the UT-B (Slc14A1) gene encodes a single isoform. In the past year, significant progress has been made in understanding the regulation of urea-transporter protein abundance in kidney, studies of genetically engineered mice that lack a urea transporter, identification of urea transporters outside of the kidney, cloning of urea transporters in nonmammalian species, and active urea transport in microorganisms. Recent findings UT-A1 protein abundance is increased by 12 days of vasopressin, but not by 5 days. Analysis of the UT-A1 promoter suggests that vasopressin increases UT-A1 indirectly following a direct effect to increase the transcription of other genes, such as the Na+-K+-2Cl− cotransporter NKCC2/BSC1 and the aquaporin (AQP) 2 water channel, that begin to increase inner medullary osmolality. UT-A1 protein abundance is also increased by adrenalectomy, and is decreased by glucocorticoids or mineralocorticoids. However, each hormone works through its own receptor. Knockout mice that lack UT-A1 and UT-A3, or lack UT-B, have a urine-concentrating defect and a decrease in inner medullary interstitial urea content. Summary Urea transporters play a critical role in the urine-concentrating mechanism. Their abundance is regulated by vasopressin, glucocorticoids, and mineralocorticoids. These regulatory mechanisms may be important in disease states such as diabetes because changes in urea-transporter abundance in diabetic rats require glucocorticoids and vasopressin.

BACKGROUND & AIMS: Urea is a major nitrogen source for commensal bacteria that inhabit the large intestine. UT-A urea transporters mediate urea movement across plasma membranes. The aim of this study was to determine whether UT-A proteins are expressed in the mouse colon and, if so, whether they have a functional role in transcellular urea transport.METHODS: Mouse colonic UT-A transporters were investigated with Northern blot analysis, immunoblotting, immunolocalization, and refractive light flux experiments.RESULTS: Northern blot analysis showed that 4 UT-A transcripts were present in mouse colon. Two peptide-targeted polyclonal antibodies showed the presence of UT-A immunoreactive proteins in mouse colon. Antiserum ML446 targeted to the N-terminus of mouse UT-A1 detected proteins of 34 and 48 kilodaltons. Antiserum ML194 targeted to the C-terminus of mouse UT-A1 detected proteins of 48, 75, and 100 kilodaltons. Immunolocalization studies using ML446 showed the presence of UT-A proteins in cells throughout the colonic crypts. ML194 specifically stained cells located in the proliferative and stem regions of the lower portion of colonic crypts. Differential centrifugation and immunoblotting of colonic epithelia showed that UT-A proteins were present in plasma membrane-enriched fractions. Refractive light flux experiments using colonic plasma membrane vesicles showed a significant urea flux, which was completely inhibited by the UT-A inhibitor phloretin.CONCLUSIONS: Functional UT-A transporters are expressed in the plasma membranes of mouse colon, indicating that these proteins may play a key role in host/bacterial interaction.

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.

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.

Expression of salt and urea transporters in rat kidney during cisplatin-induced polyuria.BackgroundCisplatin (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.MethodsMale 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.ResultsCP-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 α-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.ConclusionsChanges 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.

Urea is a small solute synthesized by many terrestrial organisms as part of the catabolism of protein. In mammals it is transported across cellular membranes by specific urea transporter (UT) proteins that are the products of two separate, but closely related genes, referred to as UT-A and UT-B. Three major UT-A isoforms are found in the kidney, namely UT-A1, UT-A2, and UT-A3. UT-A2 is found in the thin, descending limb of the loop of Henle, whereas UT-A1 and UT-A3 are concentrated in the inner medullary collecting duct. UT-A2 and UT-A3 effectively represent two halves of the whole UT-A gene and, when joined together by 73 hydrophilic amino acids, constitute UT-A1. A biophysical characterization of mouse UT-A2 and UT-A3 was undertaken by expression in Xenopus laevis oocytes and subsequent preparation of highly enriched plasma membrane vesicles for use in stopped-flow fluorometry. Both isoforms were found to be highly specific for urea, and did not permeate water, ammonia, or other molecules closely related to urea (formamide, acetamide, methylurea, and dimethylurea). Single transporter flux rates of 46,000 ± 10,000 and 59,000 ± 15,000 (means ± SE) urea molecules/s/channel for UT-A2 and UT-A3, respectively, were obtained. Overall, the UT-A2 and UT-A3 isoforms appear to have identical functional kinetics.

The aim of this study was to evaluate the long-term effects of cyclosporine (CsA) treatment on urinary concentration ability. Rats were treated daily for 4 wk with vehicle (VH; olive oil, 1 ml/kg sc) or CsA (15 mg/kg sc). The influence of CsA on the kidney's ability to concentrate urine was evaluated using functional parameters and expression of aquaporins (AQP1–4) and of urea transporters (UT-A-1–3, and UT-B). Plasma vasopressin levels and the associated signal pathway were evaluated, and the effect of vasopressin infusion on urine concentration was observed in VH- and CsA-treated rats. Toxic effects of CsA on tubular cells in the medulla as well as the cortex were evaluated with aldose reductase (AR), Na-K-ATPase-α1expression, and by determining the number of terminal transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells. Long-term CsA treatment increased urine volume and fractional excretion of sodium and decreased urine osmolality and free-water reabsorption compared with VH-treated rats. These functional changes were accompanied by decreases in the expression of AQP (1–4) and UT (UT-A2, -A3, and UT-B), although there was no change in AQP2 in the cortex and outer medulla and UT-A1 in the inner medulla (IM). Plasma vasopressin levels were not significantly different between two groups, but infusion of vasopressin restored CsA-induced impairment of urine concentration. cAMP levels and Gsα protein expression were significantly reduced in CsA-treated rat kidneys compared with VH-treated rat kidneys. CsA treatment decreased the expression of AR and Na-K-ATPase-α1and increased the number of TUNEL-positive renal tubular cells in both the cortex and medulla. Moreover, the number of TUNEL-positive cells correlated with AQP2 or UT-A3) expression within the IM. In conclusion, CsA treatment impairs urine-concentrating ability by decreasing AQP and UT expression. Apoptotic cell death within the IM at least partially accounts for the CsA-induced urinary concentration defect.