The knockout of urea transporter-B improves the hemorheological properties of erythrocyte.

OBJECTIVE: Urea transporter-B (UT-B), highly expressed in erythrocyte, confers specific permeability to urea, urea analogues and water. The purpose of this study was to determine the hemorheological properties of UT-B null erythrocyte using a series of biophysical techniques. METHODS AND RESULTS: The blood was taken from UT-B knockout and wild-type mice and the hemorheological parameters were measured. The UT-B inhibitor, PU-14, was used to treat the erythrocyte of wild-type mice in vitro and the deformability of the treated erythrocyte was analyzed. The results showed that UT-B knockout improves the hemorheological properties of erythrocyte, including increased erythrocyte deformation index, small deformation index, orientation index, low osmotic fragility and high electrophoretic rate. The UT-B inhibitor PU-14 had the similar effect as UT-B knockout on the deformation indices. The whole blood viscosity in UT-B knockout mice showed reduction trend as compared to wild-type mice. CONCLUSIONS: The data indicate that UT-B is involved in the regulation of hemorheology, which suggests that UT-B may be a potential therapeutic target for improving the hemorheology in some metabolic and hereditary diseases.

Phenylphthalazines as small-molecule inhibitors of urea transporter UT-B and their binding model.

AIM: Urea transporters (UT) are a family of transmembrane proteins that specifically transport urea. UT inhibitors exert diuretic activity without affecting electrolyte balance. The purpose of this study was to discover novel UT inhibitors and determine the inhibition mechanism. METHODS: The primary screening urea transporter B (UT-B) inhibitory activity was conducted in a collection of 10 000 diverse small molecules using mouse erythrocyte lysis assay. After discovering a hit with a core structure of 1-phenylamino-4-phenylphthalazin, the UT-B inhibitory activity of 160 analogs were examined with a stopped-flow light scattering assay and their structure-activity relationship (SAR) was analyzed. The inhibition mechanism was further investigated using in silico assays. RESULTS: A phenylphthalazine compound PU1424, chemically named 5-(4-((4-methoxyphenyl) amino) phthalazin-1-yl)-2-methylbenzene sulfonamide, showed potent UT-B inhibition activity, inhibited human and mouse UT-B-mediated urea transport with IC50 value of 0.02 and 0.69 µmol/L, respectively, and exerted 100% UT-B inhibition at higher concentrations. The compound PU1424 did not affect membrane urea transport in mouse erythrocytes lacking UT-B. Structure-activity analysis revealed that the analogs with methoxyl group at R4 and sulfonic amide at R2 position exhibited the highest potency inhibition activity on UT-B. Furthermore, in silico assays validated that the R4 and R2 positions of the analogs bound to the UT-B binding pocket and exerted inhibition activity on UT-B. CONCLUSION: The compound PU1424 is a novel inhibitor of both human and mouse UT-B with IC50 at submicromolar ranges. Its binding site is located at the So site of the UT-B structure.

Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling.

Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with ß-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased ß-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with ß-catenin, glycogen synthase kinase 3ß, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to ∼ 40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased ß-catenin phosphorylation and increased ß-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling complex.

Small-molecule inhibitors of urea transporters.

Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or 'urearetics,' in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done.

Thienoquinolins exert diuresis by strongly inhibiting UT-A urea transporters.

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 IC50 of 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.

Urea transporter UT-B deletion induces DNA damage and apoptosis in mouse bladder urothelium.

BACKGROUND: Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. METHODOLOGY/PRINCIPAL FINDINGS: Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. CONCLUSIONS/SIGNIFICANCE: UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders.

A novel small-molecule thienoquinolin urea transporter inhibitor acts as a potential diuretic.

Urea transporters (UTs) are a family of membrane channel proteins that are specifically permeable to urea and play an important role in intrarenal urea recycling and in urine concentration. Using an erythrocyte osmotic lysis assay, we screened a small-molecule library for inhibitors of UT-facilitated urea transport. A novel class of thienoquinolin UT-B inhibitors were identified, of which PU-14 had potent inhibition activity on human, rabbit, rat, and mouse UT-B. The half-maximal inhibitory concentration of PU-14 on rat UT-B-mediated urea transport was ∼0.8 µmol/l, and it did not affect urea transport in mouse erythrocytes lacking UT-B but inhibited UT-A-type urea transporters, with 36% inhibition at 4 µmol/l. PU-14 showed no significant cellular toxicity at concentrations up to its solubility limit of 80 µmol/l. Subcutaneous delivery of PU-14 (at 12.5, 50, and 100 mg/kg) to rats caused an increase of urine output and a decrease of the urine urea concentration and subsequent osmolality without electrolyte disturbances and liver or renal damages. This suggests that PU-14 has a diuretic effect by urea-selective diuresis. Thus, PU-14 or its analogs might be developed as a new diuretic to increase renal fluid clearance in diseases associated with water retention without causing electrolyte imbalance. PU-14 may establish 'chemical knockout' animal models to study the physiological functions of UTs.

Low molecular weight fucoidan against renal ischemia-reperfusion injury via inhibition of the MAPK signaling pathway.

BACKGROUND: Ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) in both native and transplanted kidneys. The objective of the present study was to evaluate whether low-molecular-weight fucoidan (LMWF) could attenuate renal IRI in an animal model and in vitro cell models and study the mechanisms in which LMWF protected from IRI. METHODOLOGY/PRINCIPAL FINDINGS: Male mice were subjected to right renal ischemia for 30 min and reperfusion for 24 h, or to a sham operation with left kidney removed. Kidneys undergone IR showed characteristic morphological changes, such as tubular dilatation, and brush border loss. However, LMWF significantly corrected the renal dysfunction and the abnormal levels of MPO, MDA and SOD induced by IR. LMWF also inhibited the activation of MAPK pathways, which consequently resulted in a significant decrease in the release of cytochrome c from mitochondria, ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3, and phosphorylation of p53. LMWF alleviated hypoxia-reoxygenation or CoCl(2) induced cell viability loss and ΔΨm dissipation in HK2 renal tubular epithelial cells, which indicates LMWF may result in an inhibition of the apoptosis pathway through reducing activity of MAPK pathways in a dose-dependent manner. CONCLUSIONS/SIGNIFICANCE: Our in vivo and in vitro studies show that LMWF ameliorates acute renal IRI via inhibiting MAPK signaling pathways. The data provide evidence that LMWF may serve as a potential therapeutic agent for acute renal IRI.

UT-B-deficient mice develop renal dysfunction and structural damage.

BACKGROUND: Urea transporter UT-B is the major urea transporter in erythrocytes and the descending vasa recta in the kidney. In this study, we investigated the effects of long-term UT-B deficiency on functional and structural defect in the kidney of 16-and 52-week-old UT-B-null mice. METHODS: UT-B-knockout mice were generated by targeted gene disruption and lacked UT-B protein expression in all organs. The urinary concentrating ability of mice was studied in terms of daily urine output, urine osmolality, and urine and plasma chemistries. Changes in renal morphology were evaluated by hematoxylin and eosin staining. RESULTS: The UT-B-null mice showed defective urine concentrating ability. The daily urine output in UT-B-null mice (2.5 ± 0.1 ml) was 60% higher and urine osmolality (985 ± 151 mosm) was significantly lower than that in wild-type mice (1463 ± 227 mosm). The 52-week-old UT-B-null mice exhibited polyuria after water deprivation, although urine osmolality was increased. At 52 weeks of age, over 31% of UT-B-null mice exhibited renal medullary atrophy because of severe polyuria and hydronephrosis. CONCLUSIONS: Long-term UT-B deficiency causes severe renal dysfunction and structural damage. These results demonstrate the important role of UT-B in countercurrent exchange and urine concentration.

Mice lacking urea transporter UT-B display depression-like behavior.

Urea transporter B is one of urea transporters that selectively transport urea driven by urea gradient across membrane and expressed abundantly in brain. To determine the physiological role of UT-B in brain, UT-B localization, urea concentration, tissue morphology of brain, and behavioral phenotypes were studied in UT-B heterozygous mice via UT-B null mice. UT-B mRNA was expressed in olfactory bulb, cortex, caudate nucleus, hippocampus and hypothalamus of UT-B heterozygous mice. UT-B null mice exhibited depression-like behavior, with urea accumulation, nitric oxide reduction, and selective neuronal nitric oxide synthase level increase in hippocampus. After acute urea loading, the urea level increased, NO production decreased in hippocampus from both types of mice. Moreover, urea level was higher, and NO concentration was lower consistently in UT-B null hippocampus than that in heterozygous hippocampus. In vitro, 25 mM urea inhibited NO production too. Furthermore, UT-B knockout induced a long-lasting notable decrease in regional cerebral blood flow and altered morphology, such as loss of neurons in CA3 region, swelling, and membranous myelin-like structure formation within myelinated and unmyelinated fibers in hippocampus. These results suggest that urea accumulation in the hippocampus induced by UT-B deletion can cause depression-like behavior, which possibly attribute to disturbance in NOS/NO system.

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism.

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts.

Role of vimentin in the inhibitory effects of low-molecular-weight heparin on PC-3M cell adhesion to, and migration through, endothelium.

Low-molecular-weight heparin (LMWH) has been used in cancer patients with venous thromboembolic complications, resulting in a higher survival rate and an inhibitory action on experimental metastasis. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with LMWH for 24 h. We found that the resulting HUVECs could significantly inhibit the highly metastatic human prostate cancer cell line (PC-3M) in terms of its adhesion to the endothelium and migration across the endothelium, according to scanning electron microscopy. We also determined the elevated levels of endothelial intercellular Ca(2+) concentration after the adhesion of PC-3M cells to HUVECs was greatly reduced by incubation with LMWH. Using proteomics, we surveyed the global protein changes in HUVECs after LMWH treatment and identified four down-regulated proteins that were possible isoforms of cytoskeletal vimentin intermediate filaments, cartilage-derived C-type lectin, and serine/threonine protein phosphatase 1ß (PP-1B). LMWH affected the morphology of vimentin and the expression levels of α(v) integrin and PP-1B in HUVECs bound to PC-3M cells. Vimentin assists in the adhesion of PC-3M cells, which was confirmed by short interfering RNA experiments. Furthermore, the direct binding of purified vimentin protein with LMWH was detected with surface plasmon resonance methods. However, when we used fluorescence-labeled heparin for 24 h to identify whether this binding occurred within cells, heparin was distributed principally around endothelial cells. Taken together, these findings suggest that the monoincubation of LMWH with HUVECs could inhibit PC-3M cell adhesion to, and migration through, endothelium. LMWH's regulation of vimentin plays a role in the antimetastatic action.

Erythrocyte permeability to urea and water: comparative study in rodents, ruminants, carnivores, humans, and birds.

Mammalian erythrocytes exhibit high urea permeability (P (urea)) due to UT-B expression in their cytoplasmic membrane. This high P (urea) allows fast equilibration of urea in erythrocytes during their transit in the hyperosmotic renal medulla. It also allows more urea (in addition to that in plasma) to participate in counter-current exchange between ascending and descending vasa recta, thus improving the trapping of urea in the medulla and improving urine concentrating ability. To determine if P (urea) in erythrocytes is related to diet and urine concentrating ability, we measured P (urea) in erythrocytes from 11 different mammals and 5 birds using stopped-flow light scattering. Carnivores (dog, fox, cat) exhibited high P (urea) (in x10(-5) cm/s, 5.3 ± 0.6, 3.8 ± 0.5 and 2.8 ± 0.7, respectively). In contrast, herbivores (cow, donkey, sheep) showed much lower P (urea) (0.8 ± 0.2, 0.7 ± 0.2, 1.0 ± 0.1, respectively). Erythrocyte P (urea) in human (1.1 ± 0.2), and pig (1.5 ± 0.1), the two omnivores, was intermediate. Rodents and lagomorphs (mouse, rat, rabbit) had P (urea) intermediate between carnivores and omnivores (3.3 ± 0.4, 2.5 ± 0.3 and 2.4 ± 0.3, respectively). Birds that do not excrete urea and do not express UT-B in their erythrocytes had very low values (<0.1 × 10(-5) cm/s). In contrast to P (urea), water permeability, measured simultaneously, was relatively similar in all mammals. The species differences in erythrocytes P (urea) most probably reflect adaptation to the different types of diet and resulting different needs for concentrating urea in the urine.

Curcumin inhibits renal cyst formation and enlargement in vitro by regulating intracellular signaling pathways.

Autosomal dominant polycystic kidney disease, a common inherited disease affecting about 1/1000 and 1/400 live births, is characterized by massive enlargement of fluid-filled cysts and eventually causes renal failure. The purpose of this study is to identify the inhibitory effect of curcumin on renal cyst development and to investigate the inhibitory mechanism. Madin-Darby canine kidney (MDCK) cyst model and murine embryonic kidney cyst model were used to evaluate inhibitory activity. Cell viability, proliferation, apoptosis, CFTR function and expression, and signaling pathways in MDCK cells were determined to explore the mechanism of cyst inhibition. Curcumin was found to significantly inhibit MDCK cyst development. At maximum dose curcumin caused 62% inhibition of the cyst formation (IC(50) was 0.12 µM). Curcumin slowed cyst enlargement in both MDCK cyst model and embryonic kidney cyst model with dose-response relationship. Curcumin neither induced cytotoxicity nor apoptosis in MDCK cells at <100 µM. Curcumin failed to affect the chloride transporter CFTR expression and function. Interestingly, curcumin inhibited forskolin-promoted cell proliferation and promoted the tubule formation in MDCK cells, which indicates curcumin promotes MDCK cell differentiation. Furthermore, curcumin reduced the intracellular signaling proteins Ras, B-raf, p-MEK, p-ERK, c-fos, Egr-1, but increased Raf-1 and NAB2 in MDCK cells exposed to forskolin. These results define that curcumin inhibits renal cyst formation and enlargement and suggest that curcumin might be developed as a candidate drug for polycystic kidney disease.

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes.

Although fatty acids enhance preadipocyte differentiation in the presence of adequate hormone cocktails, little is known regarding their effects in the absence of these hormones. We have now shown that palmitate, a common long-chain saturated fatty acid, induced apoptosis in both mouse 3T3-L1 and rat primary preadipocytes grown in a normal serum-containing medium. Treatment of preadipocytes with palmitate induced multiple endoplasmic reticulum (ER) stress responses, evidenced by increased protein content of CHOP and GRP78 and splicing of XBP-1 mRNA, as well as altered phosphorylation of eIF2alpha and increased phosphorylation of JNK and Erk1/2. Intriguingly, palmitate induced an early activation of Akt but diminished both Akt activation and its protein mass after prolonged incubation (>6 h). In association with these changes, palmitate reduced expression of beta-catenin and its downstream target, c-Myc and cyclin D1, two key prosurvival proteins. Overexpression of constitutively active Akt did not block the apoptotic effect of palmitate. Cotreatment with unsaturated fatty acids (oleate, linoleate) or with LiCl (a glycogen synthase kinase-3beta inhibitor) attenuated the palmitate-induced apoptosis. Subsequent analysis suggested that the unsaturated fatty acids probably counteracted palmitate by reducing, not eliminating, ER stress, whereas LiCl probably improved viability by activating the Wnt signaling pathway. Cotreatment of palmitate with a standard adipogenic hormone cocktail also abolished the apoptotic effect and promoted adipocyte differentiation. Collectively, our results suggest that palmitate causes multiple cellular stresses that may lead to apoptosis in preadipocytes in the absence of adipogenic stimuli, highlighting the importance of exogenous hormones in directing cell fate in response to increased fatty acid influx.