The effect of female hormones upon urate transport systems in the mouse kidney

Increased levels of serum urate in postmenopausal women are thought to be caused by a change in renal urate elimination associated with the loss of female hormones. In this study, we investigated the regulation of renal urate transporter expression by female hormones using ovariectomized mice with or without hormone replacement. Estradiol suppressed the protein levels of urate reabsorptive transporters urate transporter 1 and glucose transporter 9 (Urat1 and Glut9), and that of urate efflux transporter ATP-binding cassette sub-family G member 2 (Abcg2). Progesterone suppressed protein levels of sodium-coupled monocarboxylate transporter 1 (Smct1). However, neither estradiol nor progesterone influenced the respective levels of mRNA.     

Urat1-Uox double knockout mice are experimental animal models of renal hypouricemia and exercise-induced acute kidney injury

ABSTRACT

Renal hypouricemia (RHUC) is a hereditary disease characterized by a low level of plasma urate but with normal urinary urate excretion. RHUC type 1 is caused by mutations of the urate transporter URAT1 gene (SLC22A12). However, the plasma urate levels of URAT1 knockout mice are no different from those of wild-type mice. In the present study, a double knockout mouse, in which the URAT1 and uricase (Uox) genes were deleted (Urat1-Uox-DKO), were used as an experimental animal model of RHUC type 1 to investigate RHUC and excise-induced acute kidney injury (EIAKI). Mice were given a variable content of allopurinol for one week followed by HPLC measurement of urate and creatinine concentrations in spot urine and blood from the tail. The urinary excretion of urate in Urat1-Uox-DKO mice was approximately 25 times higher than those of humans. With allopurinol, the plasma urate levels of Urat1-Uox-DKO mice were lower than those of Uox-KO mice. There were no differences in the urinary urate excretions between Urat1-Uox-DKO and Uox-KO mice administered with 9 mg allopurinol /100 g feed. In the absence of allopurinol, plasma creatinine levels of some Urat1-Uox-DKO mice were higher than those of Uox-KO mice. Consequently, hypouricemia and normouricosuria may indicate that the Urat1-Uox-DKO mouse administered with allopurinol may represent a suitable animal model of RHUC type 1. Urat1-Uox-DKO mice without allopurinol exhibited acute kidney injury, thus providing additional benefit as a potential animal model for EIAKI. Finally, our data indicate that allopurinol appears to provide prophylactic effects for EIAKI.     

Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport

High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes.     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level.

Glucose transport in skeletal muscle is mediated by two distinct transporter isoforms, designated muscle/adipose glucose transporter (Glut4) and erythrocyte/HepG2/brain glucose transporter (Glut1), which differ in both abundance and membrane distribution. The present study was designed to investigate whether differences in insulin responsiveness of red and white muscle might be due to differential expression of the glucose transporter isoforms. Glucose transport, as well as Glut1 and Glut4 protein and mRNA levels, were determined in red and white portions of the quadriceps and gastrocnemius muscles of male Sprague-Dawley rats (body wt. approx. 250 g). Maximal glucose transport (in response to 100 nM-insulin) in the perfused hindlimb was 3.6 times greater in red than in white muscle. Red muscle contained approx. 5 times more total Glut4 protein and 2 times more Glut4 mRNA than white muscle, but there were no differences in the Glut1 protein or mRNA levels between the fibre types. Our data indicate that differences in responsiveness of glucose transport in specific skeletal muscle fibre types may be dependent upon the amount of Glut4 protein. Because this protein plays such an integral part in glucose transport in skeletal muscle, any impairment in its expression may play a role in insulin resistance.     

Association between intronic SNP in urate-anion exchanger gene, SLC22A12, and serum uric acid levels in Japanese

Serum uric acid levels are maintained by urate synthesis and excretion. URAT1 (coded by SLC22CA12) was recently proposed to be the major absorptive urate transporter protein in the kidney regulating blood urate levels. Because genetic background is known to affect serum urate levels, we hypothesized that genetic variations in SLC22A12 may predispose humans to hyperuricemia and gout. We investigated rs893006 polymorphism (GG, GT and TT) in SLC22A12 in a total of 326 Japanese subjects. Differences in clinical characteristics among the genotype groups were tested by the analysis of variance (ANOVA). In male subjects, mean serum uric acid levels were significantly different among the three genotypes. Levels in the GG genotype subjects were the highest, followed by those with the GT and TT genotypes. However, no differences between the groups were seen in the distributions of creatinine, Fasting plasma glucose (FPG), HbA(1c), total cholesterol, triglyceride, HDL cholesterol levels or BMI. A single nucleotide polymorphism (SNP) in the urate transporter gene SLC22CA12 was found to be associated with elevated serum uric acid levels among Japanese subjects. This SNP may be an independent genetic marker for predicting hyperuricemia.     

Estrogen Receptor β Signaling Induces Autophagy and Downregulates Glut9 Expression

Glut9 is highly expressed in the human kidney proximal convoluted tubular and plays a crucial role in the regulation of plasma urate levels. The gene effects were stronger among women. Our results show that 17-β-estradiol (E2) through ER (estrogen receptor) β downregulates Glut9 protein expression on human renal tubular epithelial cell line (HK2). Intriguingly, E2 does not affect the expression of Glut9 mRNA. ERβ is linked to PTEN, the PTEN gene negatively regulates the PI3K/AKT pathway, and the PI3K/AKT pathway inhibition may lead to autophagy. Further study indicates that ERβ may affect the expression of Glut9 though autophagy.     

Apical Voltage-Driven Urate Efflux Transporter NPT4 in Renal Proximal Tubule

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).     

Apical voltage-driven urate efflux transporter NPT4 in renal proximal tubule

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Increased expression of SLC2A9 decreases urate excretion from the kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Estrogen augments glucose transporter and IGF1 expression in primate cerebral cortex.

Estrogen has many positive effects on neural tissue in experimental model systems, including stimulation of neurite growth and neurotransmitter synthesis and protection against diverse types of neural injury. In humans, estrogen treatment is reputed to protect against Alzheimer's disease. To investigate potential mediators of estrogen's action and determine whether selective estrogen receptor modulators (SERMs) such as tamoxifen have estrogen-like effects in the primate brain, we evaluated the expression of glucose transporters and insulin-like growth factor 1 (IGF1) and its receptor in the frontal cortex of ovariectomized rhesus monkeys. We treated one group for 3 days with vehicle, another with 17 beta estradiol (E2), and a third with tamoxifen. The expression of facilitative glucose transporters (Gluts) 1, 3, and 4 was investigated using in situ hybridization, immunohistochemistry, and immunoblot analysis. Gluts 3 and 4 were concentrated in cortical neurons and Glut1 in capillaries and glial cells. E2 treatment induced two- to fourfold increases in Glut3 and Glut4 mRNA levels and lesser but significant increases in Glut3 and 4 protein levels. E2 treatment induced an approximately 70% increase in parenchymal Glut1 mRNA levels, but did not appreciably affect vascular Glut1 gene expression. IGF1 and IGF1 receptor mRNAs were concentrated in cortical neurons in a distribution similar to Gluts 3 and 4. IGF1 mRNA levels were significantly increased in E2-treated animals but IGF1 receptor mRNA levels were not altered by hormone treatment. Tamoxifen increased cerebral cortical Glut3 and 4 mRNA levels, but did not affect Glut1, IGF1, or IGF1 receptor expression. This study provides novel data showing that Gluts 3 and 4 and IGF1 are coexpressed by primate cerebral cortical neurons, where their expression is enhanced by estrogen. These findings suggest that up-regulation of glucose transporter and IGF1 expression may contribute to estrogen's salutary effects on neural tissue. Tamoxifen, an antiestrogen at the breast, is shown to have estrogen-like effects on higher brain centers in the monkey, suggesting that some SERMs may share estrogen's neuroprotective potential for menopausal women.     

Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses

Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.     

Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K(m): 365+/-42 microm). The transport was Na(+)-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; urate [corrected] is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).     

ABCG2 Dysfunction Increases the Risk of Renal Overload Hyperuricemia

ATP-binding cassette transporter, sub-family G, member 2 (ABCG2/BCRP) is identified as a high-capacity urate exporter, and its dysfunction has an association with serum uric acid levels and gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate “overproduction type,” “underexcretion type,” and “combined type” based on only renal urate excretion, without considering an extra-renal pathway such as gut excretion. In this study, we investigated the effects of ABCG2 dysfunction on human urate handling and the mechanism of hyperuricemia.

Clinical parameters for urate handling including urinary urate excretion (UUE) were examined in 644 Japanese male outpatients with hyperuricemia. The severity of their ABCG2 dysfunction was estimated by genotype combination of two common ABCG2 variants, nonfunctional Q126X (rs72552713) and half-functional Q141K (rs2231142).

Contrary to the general understanding that ABCG2 dysfunction leads to decreased renal urate excretion, UUE was significantly increased by ABCG2 dysfunction (P = 3.60 × 10^?10). Mild, moderate, and severe ABCG2 dysfunctions significantly raised the risk of “overproduction” hyperuricemia including overproduction type and combined type, conferring risk ratios of 1.36, 1.66, and 2.35, respectively.

The present results suggest that common dysfunctional variants of ABCG2 decrease extra-renal urate excretion including gut excretion and cause hyperuricemia. Thus, “overproduction type” in the current concept of hyperuricemia should be renamed “renal overload type,” which is caused by two different mechanisms, “extra-renal urate underexcretion” and genuine “urate overproduction.”

Our new concept will lead to a more accurate diagnosis and more effective therapeutic strategy for hyperuricemia and gout.     

Cytokine stimulation promotes glucose uptake via phosphatidylinositol-3 kinase/Akt regulation of Glut1 activity and trafficking

Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.     

铁观音茶水提物对小鼠高尿酸血症的缓解作用

近年来,高尿酸血症（hyperuricemia,HUA）在人群中频发,危害性强,并发症多。为了探究日常饮品--半发酵茶铁观音茶水对于缓解HUA是否具有辅助作用,以小鼠为实验对象,采用氧嗪酸钾和次黄嘌呤联合法构建小鼠高尿酸血症模型,21只模型鼠随机分为模型组、铁观音茶水提物组、阳性药物组,7只非模型鼠作为对照。做不同处理2周后,取小鼠血清及肾、肝、小肠。观察各器官病理变化,从细胞层面鉴定铁观音茶水提物对高尿酸血症小鼠各脏器的影响;测定与HUA相关度较高的生化指标：血清尿酸（uric acid,UA）浓度、肝黄嘌呤氧化酶（xanthine oxidase,XOD）活力,以明确造模是否成功以及判断铁观音茶水提物对HUA是否具有缓解作用;利用qRT-PCR检测尿酸合成和排泄相关基因的mRNA表达水平,并利用Western blot检测肝XOD蛋白的表达水平,以从分子层面明确铁观音茶水提物对HUA的影响。病理切片显示,相比于阳性药物组,铁观音茶水提物组小鼠的肾和肝损害程度较轻;生化指标测定结果显示,铁观音茶水提物可降低血清尿酸水平,并且抑制XOD活性;从分子层面可以看出,铁观音茶水提物显著升高了尿酸重吸收转运体尿酸转运蛋白1(uric acid transporter 1,URAT1)和有机阴离子转运蛋白3(organic anion transporter 3,OAT3)的mRNA表达水平（P<0.05）,显著降低了葡萄糖转运子9（glucose transporter 9,GLUT9）和有机阴离子转运蛋白1(organic anion transporter 1,OAT1)的表达水平（P<0.05）。虽然qRT-PCR和Western blot提示,XOD的mRNA和蛋白质的表达水平升高,但尿酸生成量却下降,推测可能是铁观音茶水提物中的某种成分使得无催化活性的XOD蛋白表达增加进而对XOD基因的转录表达造成一种正反馈。研究提示,铁观音茶水提物对小鼠高尿酸血症具有缓解作用,其缓解高尿酸血症的作用与抑制XOD活性、干预尿酸生成过程和刺激或抑制相关阴离子转运体mRNA的表达相关。