Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway

Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by Nω-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension.

Urea transporters (UTs) are a family of membrane proteins that selectively transport urea driven by urea gradient across cell membrane 1 . Two mammalian UT subfamilies have been identified. UT-A subfamily has 6 members (UT-A1~UT-A6), most of which are expressed in kidney and play an important role in urine concentrating mechanism 2 . UT-B subfamily has only one member, which has a discrete tissue distribution with high expression in erythrocyte 3 , renal descending vasa recta (DVR) 4 , brain astrocyte 5 , testis Sertoli cell 6 , urothelial cell in bladder and ureter 7,8 , endothelial cell in blood vessel 9 , etc., suggesting that UT-B may have distinct function specific to its tissue localization.
The UT-B or UT-A null mouse models were generated by gene targeting strategy in order to understand their physiological significance. Phenotypic analysis of knockout mice lacking UT-B 10 or UT-As 11 has provided evidence for the involvement of UTs in urinary concentration. Functional deletion of UT-B or UT-A isoforms resulted in significant polyuria and a urea-selective reduction in urine concentrating ability 12 , without overt abnormalities in their main biological functions, behavior, and sensory activity. However, deletion of UT-B or UT-As did not affect glomerular filtration rate (GFR) or the clearance rate of the principal solutes (Na + , K + , Cl − ) in urine, except for urea 12 . Therefore, these findings suggest that UTs might be useful as novel diuretic targets to excrete water without disturbing electrolyte balance.
For over half a century, diuretics have been used as the first-line antihypertensive drugs, which reduce cardiovascular and cerebrovascular events in hypertensive patients 13 . However, the long-term use of common diuretics, such as hydrochlorothiazide (HCTZ), can cause electrolyte imbalance, high blood glucose, hyperlipidemia, and hyperuricemia 14,15 . Therefore, UT inhibitors as diuretics used for the treatment of hypertension should have unique advantages compared with other classes of diuretics.
We have recently described that a novel UT-B inhibitor, thienoquinolin (PU- 14), is a potent inhibitor of UT-B-mediated transmembrane urea transport in human, rabbit, rat and mouse with respective IC 50 of 1.72, 1.79, 3.51 and 5.19 μ mol/L 16 . PU-14 increased urine output and decreased urine osmolality without causing electrolyte disturbance or metabolic changes 16 , suggesting that PU-14 is a potential diuretic through urea-selective diuresis. In addition, PU-14 can serve as a useful tool to explore the physiological role of UT-B in animal models.
Vasodilatation is another mechanism for diuretics, such as indapamide and HCTZ, to lower blood pressure 17 . UT-B is abundantly expressed in vascular endothelial cells 9 , which might explain its wide distribution in many extrarenal tissues. Previous study showed that UT-B deletion increased nitric oxide (NO) production via L-arginine-nitric oxide synthase (NOS)-NO pathway in mouse bladder 18 , indicating that pharmacological inhibition of UT-B can affect NO production in vascular endothelial cells and vasodilatation.
This study aimed to examine the physiological role of UT-B in blood pressure regulation using UT-B null mice and UT-B selective inhibitor PU-14, and to explore the possible mechanisms. The present results suggest that UT-B is involved in regulation of vascular function and blood pressure. The main mechanism involves the up-regulation of L-arginine-endothelial nitric oxide synthase (eNOS)-NO pathway.

UT-B deletion lowers blood pressure and augments vasodilatation in mice.
Immunofluorescence showed that UT-B was expressed in vascular endothelial cells of thoracic aortas from wild-type mice but not from UT-B null mice (Fig. 1A). Morphological analysis with hematoxylin and eosin stain showed no structural abnormality in UT-B null mouse aortas (Fig. 1B).

Inhibition of UT-B enhances eNOS-NO pathway.
Western blot analysis of thoracic aortas showed that levels of endothelial nitric oxide synthase (eNOS) and p-eNOS (ser 1177 ) were increased whereas arginase I expression was decreased in UT-B null mice, with no change of arginase II expression (Fig. 5A). In addition, the levels of eNOS and p-eNOS (ser 1177 ) were elevated in aortas from PU-14-treated WKYs and SHRs with no changes in those from vehicle groups (Fig. 5B,C).

UT-B is involved in the regulation of intracellular signaling. Immunofluorescence showed that UT-B
is expressed in bovine aortic endothelial cells (BAECs, Fig. 6A) and PU-14 showed no cytotoxicity at concentration < 50 μ mol/L (data not shown). Urea at <300 mmol/L did not affect cell viability 18 . To determine whether urea has any impact on NO production in vitro, BAECs were exposed to 25 mmol/L urea for 3 hours 9,19 , using 25 mmol/L glucose as an equal osmolality control. Urea incubation significantly increased NO concentration in culture medium (Fig. 6B). PU-14 (10 μ mol/L) significantly increased NO production, and co-incubation of urea and PU-14 produced a greater amount of NO than urea or PU-14 alone (Fig. 6B). The NO-increasing effect of urea and PU-14 was inhibited by L-NAME. Urea in the absence and presence of PU-14 elevated the expression of eNOS and reduced the expression of arginase I (Fig. 6C).

Discussion
The original objective of this study was to determine if pharmacological inhibition of urea transporter would lower blood pressure through diuresis. It is found that both UT-B knockout in mice and UT-B inhibitor PU-14-treated WKYs and SHRs had reduced blood pressure. Previous studies showed that the antihypertensive effects of diuretics result from a moderate reduction in extracellular fluid volume and plasma volume, as reflected by the loss of body weight 20 . The present study showed that plasma antidiuretic hormone level, an index of blood volume, is  comparable between wild-type and UT-B null mice. In addition, continuous PU-14 treatment did not change body weight in rats, indicating that little blood volume loss occurs after PU-14 treatment. PU-14 exerted a weak diuretic effect in SHRs, but its antihypertensive effect was stronger after seven-day PU-14 treatment. These observations led us to propose that the diuretic effect of PU-14 may have little impact on its blood pressure-lowering effect and the antihypertensive effect of PU-14 is mainly induced by vasodilator effect, with diuresis as a minor contributor.
Many studies have suggested other mechanisms underlying actions of some classical diuretics, such as HCTZ and indapamide, clinically used as the first-line antihypertensive drugs to lower blood pressure through vasodilatation 17,21-23 . The present results show that UT-B deletion and PU-14 treatment reduced blood pressure and caused relaxations of aortas and mesenteric arteries from both mice and rats. These results indicate that UT-B functional inhibition lowers blood pressure probably by vasorelaxation.
Several vasoactive substances, including NO, prostacyclin, angiotensin II, thromboxane A 2 (TXA 2 ) and endothelin-1 (ET-1) are known to participate in vascular homeostasis and blood pressure regulation. NO plays a critical role in regulation of vascular tone, control of blood flow and maintenance of vascular integrity in physiological or pathophysiological conditions 24,25 . As lowered blood pressure and enhanced vasorelaxation were observed in UT-B null mice, we propose that the eNOS-NO pathway may play a key role for UT-B to regulate blood pressure.
The work of Wanger et al. provided an important cue for us to focus on the L-arginine-eNOS-NO axis 9,19 . Though we failed to determine the L-arginine levels in plasma, vascular tissue or BAECs by HPLC, the present results suggest that UT-B functional inhibition decreased blood pressure by activating L-arginine-eNOS-NO pathway. First, ACh-induced relaxations were augmented in aortas of UT-B null mice compared with wild-type mice. Second, PU-14-induced relaxations depended on the presence of endothelium and the relaxations were blocked by NOS inhibitor L-NAME, suggesting that endothelium-derived NO mediates vasorelaxation caused by UT-B inhibition. The activity of eNOS controls NO production in blood vessels 26 . We observed that the expression and phosphorylation levels of eNOS were increased in aortas from UT-B null mice and PU-14-treated WKYs and SHRs. Although we do not know the detailed mechanisms leading to eNOS phosphorylation, the present study at least provides evidence that both short-term and long-term vascular effect of UT-B inhibition is associated with increased phosphorylation and expression of eNOS in arteries.
We next attempted to explore how UT-B deletion or functional inhibition may activate eNOS-NO pathway. Treatment of endothelial cells with urea (6.25, 12.5, 25 mmol/L) plus PU-14 (10 μ mol/L) to mimic the condition in UT-B null mice with high plasma urea level. We found that intracellular urea accumulation increased with the treatment of increasing concentration of PU-14, accompanied by the increased levels of eNOS and p-eNOS and down-regulation of arginase I.
As UT-B inhibition caused intracellular urea accumulation, there might exist a debate whether urea can be toxic in endothelial cells. Our observation revealed that plasma urea of UT-B null mice and PU-14 treated rats were both much lower than uremia (25 mmol/L) 19 , and urea did not induce obvious cytotoxicity in BAECs under 50 mmol/L (data not shown). Moreover, previous reports indicate that the accumulation of ions and toxic substances in body fluids causes uremic symptoms. And even a prolonged increase in the concentration of urea does not produce toxic reactions, at least in patients with normal kidney function 27 .
In vascular endothelial cells, two metabolic products are derived from L-arginine. One is NO catalyzed via eNOS 28 , and the other is urea catalyzed via arginase 29 . Previous studies demonstrated a beneficial effect of acute and chronic L-arginine supplementation to augment endothelium-derived NO production and thus endothelial function 30,31 . L-arginine lowers blood pressure in experimental hypertension 32,33 . Arginase is a critical regulator of NO formation by competing with NOS for their common substrate L-arginine 34 . The most direct evidence for arginase to regulate NO synthesis is that endothelial cells with stable overexpression of arginase I or arginase II decrease intracellular arginine by 25% or 11%, and reduce NO synthesis by 60% or 47%, respectively 29 . Thus, less arginase consumes less L-arginine and makes more L-arginine available for NO generation. However, an "L-arginine paradox" makes L-arginine transport, metabolism and NO synthesis even more complex. Under physiological conditions, the intracellular L-arginine level far exceeds the K m of NO synthase for L-arginine, but supplement of exogenous L-arginine still increases NO production 35,36 . Although there are explanations for this paradox, no definite mechanism has been defined. Our study focuses more on the competitive relationship between these two metabolic pathways involving L-arginine. When UT-B is defected or functionally inhibited, the intracellular urea is accumulated to exert a feedback inhibition of arginase and to elevate the activity and expression of eNOS. As the affinity of L-arginine for eNOS is considerably greater than that for arginases 37,38 , increased activity and blots for eNOS, arginase I and arginase II protein expression at different culture conditions. G: 25 mmol/L glucose; U: 25 mmol/L urea; U + D: 25 mmol/L urea + 0.1% DMSO; U + P: 25 mmol/L urea + 10 μ mol/L PU-14; U + P + L: 25 mmol/L urea + 10 μ mol/L/L PU-14 + 100 μ mol/L L-NAME. (right) Bar graph shows the density ratio of eNOS, arginase I and arginase II to β -actin. Data are mean ± SEM (n = 3).*P < 0.05, **P < 0.01 compared with G; # P < 0.05, ## P < 0.01compared with U + P (ANOVA). (D) (up)Western blots for eNOS, p-eNOS (ser 1177 ), arginase I and arginase II protein expression at different culture conditions. C: no treatment; D: 0.1% DMSO; G: 25 mmol/L glucose; U6. 25 expression of eNOS induces elevated NO production (Fig. 8). However, confirmation of this hypothesis, especially the relationship between UT-B and these two competitive metabolism pathways of L-arginine, needs further study.
However, there was only an increasing trend of plasma NO level in UT-B null mice. It was reported that the plasma nitrate level might not be a reliable estimate of endogenous NO synthesis in vascular endothelial cells, because of unmeasurable confounding impact of nitrate derived from exogenous diet or nitrate-containing drugs [39][40][41] . Although NO is the primary endothelium-derived relaxing factor, other factors may be also involved in the regulation of vascular reactivity in smaller blood vessels and blood pressure 42 . We observed increase in plasma prostacyclin and decrease in plasma angiotensin II in UT-B null mice. As it has been demonstrated that cyclooxygenase-prostacyclin pathway made contribution to vasodilatation 43,44 , we speculate that UT-B might involve in the regulation of this pathway for unknown mechanism to make vessels relax besides eNOS-NO pathway. And other studies show close relationship between angiotensin II and blood pressure for complex factors 45 . Therefore, the present study cannot discount the contribution of these factors to beneficial effects of UT-B inhibition on endothelial function for unclear reasons 46 .
Endothelial dysfunction and reduced NO bioactivity represent prominent pathophysiological abnormalities associated with hypertensive disease 47 , though the underlying mechanism is complex. Previous studies demonstrated that endothelium-or NO-dependent vasorelaxation induced by ACh is blunted in adult SHR (12 weeks old) with developed hypertension 48 . We also found that aortas from 12 weeks old SHRs showed weaker relaxing response to ACh than that from 12-week WKYs, suggesting impaired endothelial function in 12-week SHRs. But PU-14 induced similar endothelium-dependent relaxations of aortas from both 12 weeks old SHRs and WKYs, which was consistent with the increased expression and phosphorylation of eNOS in aortas from SHRs and WKYs treated with PU-14.
In summary, the present study provides novel evidence that UT-B plays an important role in regulating vascular function and blood pressure. UT-B inhibition causes intracellular urea accumulation in endothelial cells, which decreases the arginase expression and thus increases eNOS activity and expression to produce more NO, leading to endothelium-dependent vasorelaxations. Taken together, UT-B may be a novel target for the treatment of hypertension, and UT-B inhibitors could be developed as potential antihypertensive agents.

Materials and Methods
Animals. Male UT-B null mice at age of 8 weeks, with a C57BL/6J genetic background were generated as described previously 10 . Male Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at 8 weeks and 12 weeks old were supplied by Peking University Health Science Center (PUHSC) Laboratory Animal Service Center. All animals were housed at room temperature (23 ± 1 °C) and relative humidity (50%) under a regular light/dark cycle with free access to food and water. All animal experiments were conformed to the Guide for the

Effect of PU-14 on WKYs and SHRs. Adult WKYs and SHRs were adapted in metabolic cages (Harvard
Apparatus, Holliston, MA, USA) for 3 days before a seven-day treatment. After rat bladder was emptied by gentle abdominal massage, urine was collected by spontaneous voiding every 24 hours. PU-14 (PU-14 was home synthesized with purity at 99% as determined by HPLC) at 50 mg/kg in 40% (g/ml) 2-hydroxypropyl-β -cyclodextrin was subcutaneously injected every 6 hours (0:30 a.m., 6:30 a.m., 12:30 p.m., and 6:30 p.m.) for one week 16 . 40% 2-hydroxypropyl-β -cyclodextrin was used as a vehicle control. Two hours after the last administration, blood sample and thoracic aorta were collected under anesthesia with pentobarbital (1%) at 40 mg/kg body weight. The adequacy of anaesthesia was monitored based on the disappearance of the pedal with drawal reflex response to foot pinch. Urinary volume was measured by gravimetry, assuming a density of 1 g/ml. Blood pressure measurement. Blood pressure was measured using a computerized tail cuff system (Kent Scientific Corporation, Torrington, CT, USA) with a photoelectric sensor. The animals were trained for 7 days before starting the measurement to prevent stress and were prewarmed to 30-32 °C with a far infrared warming pad (DCT-25, Kent Scientific Corporation, Torrington, CT, USA). Blood pressure was recorded daily at 8:30 to 10:30 a.m. and averaged from five consecutive recordings.
Vasoreactivity measurement. Vasoreactivity assay was performed 49,50 . Animals were sacrificed by CO 2 gas inhalation and bled rapidly by cutting the carotid arteries. The thoracic aortas and main mesenteric arteries were carefully isolated and placed in cold Kreb's solution. Kreb's solution contained (in mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1 MgCl 2 , 25 NaHCO 3 , 1.2 KH 2 PO 4 and 11 D-glucose 51 . Each artery was cut into 3-5 mm rings. Rings were held in place by means of two stiff tungsten wires (diameters, 30 μ m) that were carefully passed through the lumen and fastened to clamps attached to a force transducer (Grass Instrument Co., Quincy, MA, USA) and to a micromanipulator in wire myograph. Each ring was allowed to stabilize for 60 minutes before the start of each experiment. KCl (12.5, 25, 50, 100 mmol/L) and phenylephrine (PE, Sigma, 1 nmol/L-100 μ mol/L) were used to measure vessel contractility. The rings were pre-constricted with PE (1 μ mol/L), and the relaxation was measured in response to cumulative concentrations of acetylcholine (ACh, Sigma, 1 nmol/L-10 μ mol/L), PU-14 (0.01-30 μ mol/L) or sodium nitroprusside (SNP, Sigma, 1 nmol/L-100 μ mol/L ). Some arterial rings were subjected to 30 minutes exposure to L-NAME (Sigma, 100 μ mol/L, nitric oxide synthase inhibitor) and then endothelium-dependent relaxations in response to cumulative additions of ACh were measured. The data were analyzed with a PowerLabData Acquisition Systemand LabChart pro software (AD Instruments, Colorado Springs, CO, USA). NO assay. Blood samples were centrifuged at 5,000 rpm for 15 minutes and serum was collected. BAECs were treated at 80% confluence and cell culture medium was obtained after treatment for 3 hours 9,19 . Nitric oxide (NO) concentration in the plasma and cell culture medium was measured by conversion of nitric oxide to nitrate and nitrite using an NO assay kit (Jiancheng Bioengineering Co., Nanjing, Jiangsu, China). Intracellular urea accumulation assay. BAECs at 80% confluence were given different treatment. The cell samples were collected, homogenized in 100 μ l of distilled water and centrifuged at 12,000 rpm for 20 minutes at 4 °C and the supernatant was for urea measurement using QuantiChrom Urea Assay kit (Roche Diagnostics, Indianapolis, IN, USA). Results were expressed relative to protein content.
Biochemical assay. Vasoactive factors including prostacyclin, thromboxane A 2 (TXA 2 ), endothelin-1 (ET-1), angiotensin II, antidiuretic hormone (ADH) and aldosterone in plasma were assayed by radioimmunoassay kit (Beijing Northern Instrument of Biological Technology, Beijing, China). Sodium, potassium, chloride and creatinine were measured in a clinical chemistry laboratory of Peking University Third Hospital.
Cell culture. Bovine aortic endothelial cells (BAECs, a gift from Dr. Xian Wang, Peking University Health Science Center, Beijing, China) were cultured at 37 °C in a humidified 95% air/5% CO 2 atmosphere in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA ) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells used in this study were from passages 3 to 8.
Western blot Analysis. Arteries or cells were placed into RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein samples (30 μ g) were separated with 7.5% SDS-PAGE