Molecular approaches to urea transporters

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

Regulation of renal urea transporters

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

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.     

Down-regulation of urea transporters in the renal inner medulla of lithium-fed rats

BACKGROUND: Lithium is commonly used to treat bipolar psychiatric disorders but can cause reduced urine concentrating ability. METHODS: To test whether lithium alters UT-A1 or UT-B urea transporter protein abundance or UT-A1 phosphorylation, rats were fed a standard diet supplemented with LiCl for 10 or 25 days, and then compared to pair-fed control rats. To investigate another potential mechanism for decreased urea transport, inner medullary collecting duct (IMCD) suspensions from lithium-fed or control rats were incubated with 32P-orthophosphate to measure the phosphorylation of UT-A1. RESULTS: In lithium-fed rats (25 days), UT-A1 abundance was reduced to 50% of control rats in IM tip and to 25% in IM base, and UT-B abundance was reduced to 40% in IM base. Aquaporin-2 (AQP2) protein abundance was reduced in both IM regions. Vasopressin (100 pmol/L) increased UT-A1 phosphorylation in IMCD suspensions from control but not from lithium-fed rats; a higher vasopressin concentration (100 nmol/L) increased UT-A1 phosphorylation in control and lithium-fed rats. CONCLUSIONS: Decreases in UT-A1, UT-B, and AQP2 protein abundance, and/or vasopressin-stimulated phosphorylation of UT-A1, can contribute to the reduced urine concentrating ability that occurs in lithium-treated rats.     

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

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

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

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

Renal transplantation modulates expression and function of receptors and transporters of rat proximal tubules

Kidney transplantation often leads to disturbances of solute and volume maintenance in humans. To investigate underlying mechanisms, expression and function of renal transporters and receptors of the proximal tubule (PT) were analyzed in an acute rejection model of rat kidney transplantation. Semiquantitative RT-PCR and Western blot, histology, immunohistochemistry, and microfluorometry were performed on whole kidneys and isolated PT. With acute rejection, Na+/H+-exchanger type-3 (NHE-3) was markedly downregulated. Na+-HCO(3)(-)-cotransporter (NBC-1) and Na+-glucose transporter type-2 (SGLT2) were upregulated after transplantation. Expressions of Na+/H+-exchanger type-1 (NHE-1), Na+/K+-ATPase (NKA), angiotensin II (AngII) receptor (AT-1), or natriuretic peptide receptor (GC-A) were unaltered. Microfluorometric analyses of intracellular pH, Na+, and Ca2+ demonstrated a decrease in NHE-3 function and AngII-mediated stimulation of NHE-3. AngII-mediated inhibition of NHE-1 and function of all other transporters tested remained unaltered. Function of AT-1 and GC-A were unaffected. Reduced expression of NHE-3 was also confirmed by semiquantitative immunohistochemistry. These findings suggest that expression and function of transmembrane proteins involved in Na+-transport after transplantation and rejection is specifically modulated. The local renin-angiotensin-system is apparently not altered. Downregulation of NHE-3 may be a protective mechanism occurring in the graft.     

Renal handling of urea in subjects with persistent azotemia and normal renal function

Fourteen subjects with persistent azotemia and normal glomerular filtration rate were studied by renal clearances and hormonal determinations to establish the nephron site of altered urea transport and the mechanism(s) responsible for their azotemia. During constant alimentary protein, urea nitrogen appearance was normal and urea clearance was much lower than in 10 age-matched control subjects (23.3 +/- 2.1 ml/min and 49.6 +/- 2.6 ml/min per 1.73 m2, P less than 0.001). Inulin and para-aminohippurate clearances, blood volume and plasma concentration of antidiuretic hormone were within normal limits. During maximal antidiuresis, in spite of greater urea filtered load, the urinary excretion of urea was less, and both the maximum urinary osmolality and the free-water reabsorption relative to osmolar clearance per unit of GFR were greater than in control subjects. After sustained water diuresis, the plasma urea concentration markedly decreased to near normal levels in azotemic subjects. The basal urinary excretion of prostaglandins E2 was significantly reduced in azotemic subjects and was directly correlated with fractional urea clearance (r = 0.857, P less than 0.001). An additional group of control subjects (N = 8) showed a marked reduction of fractional clearance of urea after inhibition of prostaglandin synthesis (P less than 0.01). These data suggest that azotemia is due to increased tubular reabsorption of urea in the distal part of nephron, presumably because of increased back diffusion in the papillary collecting duct, accounting for the enhanced maximum urinary osmolality and free-water reabsorption. Renal prostaglandin E2 may participate in the pathogenesis of azotemia by altering recycling of urea in the medulla.     

氨转运蛋白Rh b型糖蛋白在低蛋白饮食大鼠肾脏的表达

目的 研究低蛋白饮食对氨转运蛋白Rh b型糖蛋白（Rhbg ）在大鼠肾脏表达的影响。 方法 利用Western印迹分别检测Rhbg在低蛋白饮食组和正常对照组大鼠肾脏皮质、内髓、外髓的表达。利用单标记免疫组化法、双标记免疫组化法和定量免疫组化法检测Rhbg在两组中肾小管主细胞和暗细胞的表达。 结果 与正常对照组比较,在大鼠肾脏皮质,低蛋白饮食组Rhbg 的蛋白水平显著增高（954778±509288比275701±262374,P < 0.05）;在大鼠肾脏内髓和外髓,低蛋白饮食组Rhbg 的蛋白水平无明显变化;在大鼠肾脏皮质集合管主细胞,低蛋白饮食组免疫组化Rhbg表达像素值（1310±357）显著高于对照组（896±154,P < 0.05）;在大鼠肾脏皮质集合管暗细胞,低蛋白饮食组免疫组化Rhbg表达像素值（1550±497）显著高于对照组（926±251,P < 0.05）;在大鼠肾脏内髓集合管和外髓集合管的主细胞或暗细胞中,两组间免疫组化Rhbg表达无显著差异。 结论 饮食中蛋白的限制可能会增加Rhbg在大鼠肾脏皮质集合管的主细胞和暗细胞的表达。     

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。     

Glucose transporters in diabetic nephropathy

Changes in glucose transporter expression in glomerular cells occur early in diabetes. These changes, especially the GLUT1 increase in mesangial cells, appear to play a pathogenic role in the development of ECM expansion and perhaps other features of diabetic nephropathy. In addition, it appears that at least some diabetic patients may be predisposed to nephropathy because of polymorphisms in their GLUT1 genes. GLUT1 overexpression leads to increased glucose metabolic flux which in turn triggers the polyol pathway and activation of PKC and 1. Activation of these PKC isoforms can lead directly to AP-1 induced increases in fibronectin expression and ECM accumulation. Other, more novel effects of GLUT1 on cellular hypertrophy and injury could also promote changes of diabetic nephropathy. Strategies to prevent GLUT1 overexpression could ameliorate or prevent the progression of diabetic nephropathy.     

葡萄糖转运蛋白与脑缺氧缺血

葡萄糖转运蛋白是哺乳动物细胞转运葡萄糖的主要载体,介导葡萄糖通过易化扩散由细胞膜外转运至细胞内,葡萄糖转运蛋白(glucose transporter,GLUT)1和GLUT3是脑内负责葡萄糖转运的两个重要载体.脑缺氧缺血可以增加GLUT1和GLUT3的合成,以维持脑组织能量供给.现将主要围绕脑内GLUT1和GLUT3的分布、结构、功能以及脑缺氧缺血对脑内GLUT1和GLUT3表达的影响和调控机制及意义作一综述.     

葡萄糖转运蛋白与脑缺氧缺血

葡萄糖转运蛋白是哺乳动物细胞转运葡萄糖的主要载体,介导葡萄糖通过易化扩散由细胞膜外转运至细胞内,葡萄糖转运蛋白(glucose transporter,GLUT)1和GLUT3是脑内负责葡萄糖转运的两个重要载体.脑缺氧缺血可以增加GLUT1和GLUT3的合成,以维持脑组织能量供给.现将主要围绕脑内GLUT1和GLUT3的分布、结构、功能以及脑缺氧缺血对脑内GLUT1和GLUT3表达的影响和调控机制及意义作一综述.     

Urate transporters: an evolving field

Urate (uric acid) is the end product of purine metabolism in human beings owing to the genetic loss of hepatic urate oxidase (uricase). Despite its potential advantage as an antioxidant, sustained hyperuricemia is associated with gout, renal diseases, hypertension, and cardiovascular diseases. Because the kidney plays a dominant role in maintaining serum urate levels through its excretion, it is important to understand the molecular mechanism of renal urate handling. Although molecular identification of the urate/anion exchanger URAT1 (SLC22A12) in 2002 paved the way for successive identification of several urate transport–related proteins, the entire picture of effective renal urate handling in human beings has not yet been clarified. Recently, several genome-wide association studies have revealed close associations between serum urate levels and single nucleotide polymorphisms in at least 10 genetic loci including eight transporter-related genes. These findings led us to consider the roles of urate transporters in extrarenal tissues such as the intestine. In this review, we discuss various aspects of transmembrane transport of urate in the human body.     

Quest for postdialysis urea rebound-equilibrated Kt/V with only intradialytic urea samples.

BACKGROUND: Postdialysis urea rebound (PDUR) is a cause of Kt/V overestimation when it is calculated from predialysis and the immediate postdialysis blood urea collections. Measuring PDUR requires a 30- or 60-minute postdialysis sampling, which is inconvenient. Several methods had been devised for a reasonable approach to determine PDUR-equilibrated Kt/V in short dialysis without the need for a delayed sample. The aim of our study was to compare these different Kt/V methods during the longer eight-hour hemodialysis sessions, and to determine the optimum intradialytic urea sample time that fits best with PDUR. METHODS: The study included 21 patients (mean age 71.9 years) who were hemodialyzed for 60+/-60 months at three times eight hours weekly, using bicarbonate dialysate and cellulosic membranes. Blood urea samples were obtained at onset, and then at 17, 33, 50, 66, 75, 80, 85, and 100% of the dialysis session times, after 30 seconds of low flow, and then at 60-minutes postdialysis. All patients had a meal during dialysis. We compared four different formulas of Kt/V [(a) Kt/V-Smye with a 33% dialysis time urea sample, (b) two-pool equilibrated eKt/V, (c) Kt/V-std (Daugirdas-2) obtained with an immediate postdialytic sample, and (d) the different intradialytic urea samples for Kt/V (50, 66, 75, 80, and 85% of dialysis time)] with the equilibrated 60-minute PDUR Kt/V (Kt/V-r-60) formula as the reference method. RESULTS: The mean PDUR was 17.2+/-9%, leading to an overestimation of Kt/V-std by 12.2%. Kt/V-r-60 was 1.68+/-0.34. Kt/V-std was 1.88+/-0.36 (Delta = 12.2+/-4.8%, r = 0.8). eKt/V was 1.77+/-0.3 (Delta = 5+/-5%, r = 0.96), and Kt/V-Smye was 1.79+/-0.47 (Delta = 5.2+/-14%, r = 0.9). The best time for the intradialytic sampling was 80% (that is, at 6 hr and 24 min). The Kt/V-80 was 1.64+/-0.3 and was best fitted with Kt/V-r-60 (Delta = -1.8+/-8%, r = 0.91). The mean intradialytic urea evolution showed a three-exponential rate, in discrepancy with the two-exponential rate theoretical model. CONCLUSIONS: These results confirm that a significant postdialysis rebound exists in an eight-hour dialysis. An intradialytic urea sample taken at 80% of the total session time permits an estimation of the 60-minute Kt/V-rebound without the necessity of taking a delayed sample, with better accuracy than eKt/V or especially Kt/V-Smye. This may be related to a particular urea kinetics curve on the longer dialysis duration, which needs to be studied further.     

On-line urea kinetics in haemodiafiltration

BACKGROUND.: Calculation of Kt/V and assessment of nutrition have so farbeen dependent upon off-line urea measurements of blood or dialysatesamples. Here we describe a biosensor for on-line urea measurementduring haemodiafiltration. METHODS.: The biosensor consisted of a cartridge containing covalentlylinked urease placed between two conductivity cells. The biosensorwas placed on the outlet line of a haemofilter in series witha dialyser in order to obtain an aliquot of plasma ultrafiltratefor on-line measurement of urea. RESULTS.: Urea nitrogen concentrations were highly correlated to the difference() in conductivity measured by the two conductivity cells bothin aqueous solutions (in-vitro studies, y=–6.676+32.12x,R²=0.998, P<0.0001) and in ultrafiltrates (ex-vivo studies,y=–6.7+32.01x, R²=0.98, P<0.00001). conductivity washighly reproducible (% variation: 0.8–5.3%) and stable(maximal % variation at 150 mg/dl after 180 min: 0.9±0.3vs initial values). The intradialytic plasma water urea profilewas obtained in 10 haemodialysis patients. To study recirculation,the plasma water urea profile was analysed before and 3 minafter stopping the dialysate flow. The pre- and post-stoppedflow ratio (1.21±0.1, mean±1 SD) was superimposableto conventional blood sampling data (opposite arm venous/arterial:1.22±0.11) and allowed correction for recirculation.A novel approach to urea kinetic modelling was described andused to reliably project end-dialysis and post-dialysis reboundurea concentration as early as 90 min. Projected (29.2±10.4g) or measured (29.8±10.5 g) net urea removal was highlycorrelated with the amount of urea collected in the total spentdialysate (29.7±10.6 g) (R²=0.99, R²=0.97 respectively). CONCLUSIONS.: These results indicate that on-line, real-time analysis of ureakinetics may provide information on delivery of adequate dialysisin high-efficiency techniques.