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.     

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.     

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.     

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.     

Effects of aldosterone on the protein expressions and the urea transport activity of UT-A1 and UT-A3

Objective To investigate the effects of protein expressions and the urea transport activity of aldosterone on urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) in HEK293 cells and Xenopus laevis oocytes. Methods (1) Western Blot was used to investigate the protein expressions of UT-A1 and UT-A3. (2) Cell surface biotinylation was used to investigate the protein expressions of UT-A1 and UT-A3 on the cell surface of Xenopus laevis oocytes. (3) 14C-urea transport experiment was conducted to investigate the transport activity of UT-A1 and UT-A3 in Xenopus laevis oocytes. Results (1) Compared with UT-A1 or UT-A3 high expression groups, the total protein levels of UT-A1 and UT-A3 were all significantly reduced in aldosterone treatment groups (all P＜0.01). (2) Compared with UT-A1 or UT-A3 high expression groups, the levels of protein expression on cell surface were all significantly reduced in aldosterone groups (all P＜0.01). (3) Compared with UT-A1 or UT-A3 high expression groups, 14C-urea transport experiment results showed that aldosterone treatment groups had significantly reduced the urea transporter activity of UT-A1 (1 min: 94.32±9.044 vs 40.68±4.274, P＜0.01, n=6; 3 min: 165.0±4.7 vs 80.3±0.6, P＜0.01, n=6), and UT-A3 (1 min: 204.6±3.1 vs 176.7±9.1, P＜0.05, n=6; 3 min: 371.4±14.9 vs 318.8±12.0, P＜0.05, n=6). Conclusion Aldosterone can directly down-regulate the protein expressions of UT-A1 and UT-A3 in both total protein and cell surface level, which reduces their urea transport activity.     

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.     

Lessons on renal physiology from transgenic mice lacking aquaporin water channels

Several aquaporin-type water channels are expressed in kidney: AQP1 in the proximal tubule, thin descending limb of Henle, and vasa recta; AQP2, AQP3, and AQP4 in the collecting duct; AQP6 in the papilla; and AQP7 in the proximal tubule. AQP2 is the vasopressin-regulated water channel that is important in hereditary and acquired diseases affecting urine-concentrating ability. It has been difficult to establish the roles of the other aquaporins in renal physiology because suitable aquaporin inhibitors are not available. One approach to the problem has been to generate and analyze transgenic knockout mice in which individual aquaporins have been selectively deleted by targeted gene disruption. Phenotype analysis of kidney and extrarenal function in knockout mice has been very informative in defining the role of aquaporins in organ physiology and addressing basic questions regarding the route of transepithelial water transport and the mechanism of near iso-osmolar fluid reabsorption. This article describes new renal physiologic insights revealed by phenotype analysis of aquaporin-knockout mice and the prospects for further basic and clinical developments.     

The renal adverse effects of ibuprofen are not mediated by AQP2 water channels

The aim of this study was to determine (1) whether ibuprofen treatment in very preterm infants causes an increase in the renal water channel aquaporin-2 (AQP2) activity in the collecting duct via prostaglandin synthesis inhibition and (2) whether AQP2 activity remains disturbed long after ibuprofen treatment has ended. This was a prospective study involving premature infants with a gestation age of 27–31 weeks who received treatment between December 2005 and August 2006 in a tertiary Neonatal Intensive Care Unit. Each ibuprofen-treated infant was matched to two controls. Renal glomerular and tubular function were evaluated weekly for 1 month, and urinary AQP2 was measured by immuno-dotting. In total, 166 longitudinal samples were analyzed in 36 infants. Median [interquartile range] gestational age and birthweight were 28 [27.0–29.5] weeks and 1160 [1041–1242] g, respectively. Perinatal factors were similar in both groups. Urine output was significantly decreased in the ibuprofen-treated infants during the treatment. The urinary AQP2 level decreased significantly from day 2 to day 7 in both groups and was similar thereafter for the first month of life in ibuprofen-treated and control groups. Based on our results, we conclude that ibuprofen-induced oligo-anuria is not associated with a change in AQP2 activity and that ibuprofen does not affect AQP2 activity during the first month of life in very preterm neonates.     

Activation of AQP2 water channels without vasopressin: therapeutic strategies for congenital nephrogenic diabetes insipidus

Congenital nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentrating ability. Symptomatic polyuria is present from birth, even with normal release of the antidiuretic hormone vasopressin by the pituitary. Over the last two decades, the aquaporin-2 (AQP2) gene has been cloned and the molecular mechanisms of urine concentration have been gradually elucidated. Vasopressin binds to the vasopressin type II receptor (V2R) in the renal collecting ducts and then activates AQP2 phosphorylation and trafficking to increase water reabsorption from urine. Most cases of congenital NDI are caused by loss-of-function mutations to V2R, resulting in unresponsiveness to vasopressin. In this article, we provide an overview of novel therapeutic molecules of congenital NDI that can activate AQP2 by bypassing defective V2R signaling with a particular focus on the activators of the calcium and cAMP signaling pathways.     

尿素和尿素通道在尿浓缩机制中的作用

尿液在肾脏中的浓缩过程依赖于尿素在肾髓质的积累,这种积累是通过尿素在集合管、直小血管和细降支之间的肾内循环完成的,这些部位分布有特定的尿素通道蛋白（分别称为UT—Al、UT～A3、UT—B、UT—A2）。本文将综述对UT—B基因敲除小鼠研究得到的一些新进展：①哺乳动物肾脏对尿素的处理;②UT—B缺失后对尿浓缩能力的影响;③因种属差异,小鼠、大鼠和人的肾脏对尿素进行排泄和浓缩的程度各不相同。总之,对uT—B基因敲除鼠的研究提示尿素在肾髓质中逆流倍增的循环,对总体尿浓缩能力比Henle环起到更重要的作用。     

Magnetic resonance imaging of urea transporter knockout mice shows renal pelvic abnormalities

Many transgenic and knockout mice with increased urine flow have structural abnormalities of the renal pelvis and inner medulla. Here, we used high resolution contrast enhanced T1-weighted magnetic resonance imaging of mice whose urea transporters UT-A1 and UT-A3 were deleted (UT-A1/3(-/-) mice) as a model for the in vivo study of such abnormalities. Three distinct variations in the appearance of the renal pelvis were found. These included normal kidneys with no accumulation of contrast agent in the renal pelvis; infrequent frank right-sided unilateral hydronephrosis with marked atrophy of the renal medulla; and a renal pelvic reflux pattern characterized by the presence of contrast agent in the renal pelvis surrounding the renal inner medulla but no substantial atrophy of the medulla. This last pattern was found in most of the advanced age UT-A1/3(-/-) mice and in aquaporin-1 knockout mice. The UT-A1/3(-/-) mice also had increased mean arterial blood pressures. Feeding the mice a low protein diet did not prevent development of their renal pelvic abnormalities. Our studies show that real time imaging of renal pelvic structure in genetically manipulated mice provides a tool for the non-destructive, temporal evaluation of kidney structure.     

Changes in aquaporin (AQP)2 and AQP3 expression in ovariectomized rat urinary bladder: potential implication of water permeability in urinary bladder

Purpose  

AQPs have recently been reported to be expressed in rat and human urothelium. The purpose of this study was to investigate the effect of ovariectomy on the expression of AQP2 and AQP3 in rat urothelium.     

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.     

Reduced renal expression of AQP2, p-AQP2 and AQP3 in haemorrhagic shock-induced acute renal failure.

BACKGROUND: The aims of this study were to investigate the changes in the expression levels of renal aquaporins (AQPs) in response to haemorrhagic shock (HS) in rats and whether a change in the expression of AQPs was associated with parallel changes in urinary concentration. METHODS: HS was induced by withdrawal of blood through the femoral artery in rats. A mean arterial blood pressure (MAP) of 40 mmHg was maintained for 1 h before blood was reinfused, and rats were kept in metabolic cages for urine measurements. Two days after HS, we examined the abundance of AQPs in kidney by semiquantitative immunoblotting. RESULTS: HS rats (n = 13) developed acute renal insufficiency (creatinine clearance was 5.5 +/- 0.4 vs 6.9 +/- 0.3 ml/min/kg in sham-operated rats, n = 13, P < 0.05) and decreased urine osmolality (888 +/- 88 vs 1799 +/- 110 mosmol/kg H(2)O, P < 0.05). Consistent with this, semiquantitative immunoblotting revealed that the abundance of AQP2, phosphorylated (Ser256) AQP2 (p-AQP2) and AQP3 in whole kidney was significantly decreased after 2 days to 33 +/- 4, 41 +/- 9 and 35 +/- 14% of sham levels, respectively (P < 0.05). Also, the abundance of AQP2, p-AQP2 and AQP3 in inner medulla was markedly decreased to 36 +/- 8, 39 +/- 10 and 34 +/- 16% of sham levels (P < 0.05). In contrast, the abundance of AQP1 was not significantly changed compared with sham levels. CONCLUSIONS: The expression of the collecting duct water channel AQP2, p-AQP2 and AQP3 was significantly downregulated after HS, which may play an important role in the impaired urinary concentrating ability in HS-induced acute renal failure.     

Insulin resistance and lipodystrophy in mice lacking ribosomal S6 kinase 2

The p90 ribosomal S6 kinase 2 (RSK2) is a serine/threonine kinase with high expression levels in adipose tissue. Numerous in vitro studies show that RSK2 is activated by a broad number of cellular stimuli and suggest that RSK2 is involved in the regulation of a variety of cellular processes. However, the physiological role of RSK2 still remains elusive. We therefore generated rsk2 knockout (KO) mice to better understand the function of RSK2 in vivo. Birth weights of RSK2 KO mice are normal, but the body weight is reduced with age, as compared with wild-type littermates. We found that the difference in body weight was largely caused by a specific loss of white adipose tissue that is accompanied by reduced serum levels of the adipocyte-derived peptide, leptin. KO mice also have impaired glucose tolerance and elevated fasting insulin and glucose levels that are restored following administration of low amounts of leptin, which do not affect food intake. We conclude that RSK2 plays a novel and an important role in regulation of adipose mass in mice and speculate that the reduction in fat tissue may negatively affect insulin sensitivity, as observed in human lipodystrophy, through reduced levels of adipocyte-derived factors, such as leptin.     

Upregulation of serotonin-receptor-2a and serotonin transporter expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia

Purpose

Congenital diaphragmatic hernia (CDH) is attributed to severe pulmonary hypoplasia and pulmonary hypertension (PH). PH is characterized by structural changes resulting in vascular remodeling. Serotonin, a potent vasoconstrictor, plays a central role in the development of PH. It exerts its constricting effects on the vessels via Serotonin receptor 2A (5-HT_2A) and induces pulmonary smooth muscle cell proliferation via the serotonin transporter (5-HTT). This study was designed to investigate expressions of 5-HT_2A and 5-HTT in the pulmonary vasculature of rats with nitrofen-induced CDH.

Methods

Rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen and control group (n = 32). Pulmonary RNA was extracted and mRNA level of 5HT_2A was determined by qRT-PCR. Protein expression of 5HT_2A and 5-HTT was investigated by western blotting. Confocal immunofluorescence double-staining for 5-HT_2A, 5-HTT, and alpha smooth muscle actin were performed.

Results

Pulmonary 5-HT_2A gene expression levels were significantly increased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy confirmed increased pulmonary protein expression in CDH lungs compared to controls.

Conclusion

Increased gene and protein expression of 5HT_2A and 5-HTT in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that 5HT_2A and 5-HTT are important mediators of PH in nitrofen-induced CDH.     

Delayed reepithelialization and basement membrane regeneration after wounding in mice lacking CXCR3

Wound healing is a complex, orchestrated series of biological events that is controlled by extracellular components that communicate between cell types to re-establish lost tissue. We have found that signaling by ELR-negative CXC chemokines through their common CXCR3 receptor is critical for dermal maturation during the resolving phase. In addition there needs to be complete maturation of the epidermis and regeneration of a delineating basement membrane for proper functioning. The role of this ligand–receptor system appears confounding as one ligand, CXCL4/(PF4), is present during the initial dissolution and two others, CXCL10/(IP-10) and CXCL11/(IP-9/I-TAC), are expressed by keratinocytes in the later regenerative and resolving phases during which the basement membrane is re-established. We examined CXCR3 signaling role in healing using a mouse lacking this receptor, as all three ligands act solely via the common receptor. Reepithelialization was delayed in CXCR3-deficient mice in both full and partial-thickness excisional wounds. Even at 90 days postwounding, the epidermis of these mice appeared less mature with lower levels of E-cadherin and cytokeratin 18. The underlying basement membrane, a product of both dermal fibroblasts and epidermal keratinocytes, was not fully established with persistent diffuse expression of the matrix components laminin 5, collagen IV, and collagen VII throughout the wound bed. These results suggest that CXCR3 and its ligands play an important role in the re-establishment of the basement membrane and epidermis. These studies further establish the emerging signaling network that involves the CXCR3 chemokine receptor and its ligands as a key regulator of wound repair.