Kate Witkowska

SLC2A9 Is a High-Capacity Urate Transporter in Humans

Background Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man. Methods and Findings We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82). Conclusions This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.

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 kidneys 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.

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

Background: A hydrophobic residue in TM7 of the hSLC2A9 was found to affect hexose transport. Results: Both Ile-335 and Trp-110 affect fructose trans-acceleration of urate, whereas only Trp-110 directly affects urate transport. Conclusion: hSLC2A9 handles urate/fructose transport differently. Significance: This study provides further information of how hSLC2A9 handling its substrates could be beneficial for future pharmaceutical treatments used in related diseases. 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.

A blood pressure-associated variant of the SLC39A8 gene influences cellular cadmium accumulation and toxicity

Genome-wide association studies have revealed a relationship between inter-individual variation in blood pressure and the single nucleotide polymorphism rs13107325 in the SLC39A8 gene. This gene encodes the ZIP8 protein which co-transports divalent metal cations, including heavy metal cadmium, the accumulation of which has been associated with increased blood pressure. The polymorphism results in two variants of ZIP8 with either an alanine (Ala) or a threonine (Thr) at residue 391. We investigated the functional impact of this variant on protein conformation, cadmium transport, activation of signalling pathways and cell viability in relation to blood pressure regulation. Following incubation with cadmium, higher intracellular cadmium was detected in cultured human embryonic kidney cells (HEK293) expressing heterologous ZIP8-Ala391, compared with HEK293 cells expressing heterologous ZIP8-Thr391. This Ala391-associated cadmium accumulation also increased the phosphorylation of the signal transduction molecule ERK2, activation of the transcription factor NFκB, and reduced cell viability. Similarly, vascular endothelial cells with the Ala/Ala genotype had higher intracellular cadmium concentration and lower cell viability than their Ala/Thr counterpart following cadmium exposure. These results indicate that the ZIP8 Ala391-to-Thr391 substitution has an effect on intracellular cadmium accumulation and cell toxicity, providing a potential mechanistic explanation for the association of this genetic variant with blood pressure.

The biological impact of blood pressure associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle.

Abstract Elevated blood pressure (BP) is a major global risk factor for cardiovascular disease. Genome-wide association studies have identified several genetic variants at the NPR3 locus associated with BP, but the functional impact of these variants remains to be determined. Here we confirmed, by a genome-wide association study within UK Biobank, the existence of two independent BP-related signals within NPR3 locus. Using human primary vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) from different individuals, we found that the BP-elevating alleles within one linkage disequilibrium block identified by the sentinel variant rs1173771 was associated with lower endogenous NPR3 mRNA and protein levels in VSMCs, together with reduced levels in open chromatin and nuclear protein binding. The BP-elevating alleles also increased VSMC proliferation, angiotensin II-induced calcium flux and cell contraction. However, an analogous genotype-dependent association was not observed in vascular ECs. Our study identifies novel, putative mechanisms for BP-associated variants at the NPR3 locus to elevate BP, further strengthening the case for targeting NPR-C as a therapeutic approach for hypertension and cardiovascular disease prevention.

203 Hypoxia-inducible Factor-1 Regulates Matrix Metalloproteinase-14 Expression: Underlying Effects of Hypoxia and Statins

Introduction Matrix metalloproteinase-14 (MMP14) plays an important role in atherosclerosis and angiogenesis. There is evidence indicating that hypoxia and inflammatory stimuli up-regulate MMP14 expression whereas statins have an opposite effect. However, the molecular mechanism by which these factors modulate MMP14 expression is unclear. We sought to investigate whether it involves the transcription factor hypoxia-inducible factor-1 (HIF1). Methods and Results A bioinformatics analysis of the MMP14 gene promoter identified two regions matched with the consensus sequence of HIF1-responsive element (HRE). In vitro DNA-protein interaction assay demonstrated the ability of these sequences to complex with HIF1α/HIF1β, and chromatin immunoprecipitation showed HIF1 binding to the MMP14 promoter in vascular endothelial cells under hypoxia. Cell transfection and promoter-reporter assays demonstrated that augmenting HIF1α/HIF1β expression increased MMP14 gene promoter activity and this effect was abolished by mutating the HREs in the MMP14 promoter. MMP14 mRNA and protein assays showed that hypoxia increased MMP14 expression in endothelial cells but this effect diminished in endothelial cells with HIF1α knockdown or knockout. HIF1α knockdown/knockout or MMP14 inhibition by a blocking antibody attenuated hypoxia-induced endothelial cell proliferation/migration and angiogenesis. Simvastatin reduced HIF1α in endothelial cells and mitigated hypoxia- or HIF1-induced MMP14 expression, endothelial cell proliferation/migration, and angiogenesis. Conclusions The results of this study indicate that hypoxia increases MMP14 expression in vascular endothelial cells by inducing HIF1 binding to HREs in the MMP14 gene promoter, and this action is attenuated by simvastatin.

LB03.08: HYPERTENSION RELATED VARIANT OF SOLUTE CARRIER FAMILY 39 MEMBER 8 GENE INFLUENCES CADMIUM UPTAKE AND CELL TOXICITY.

Objective: hSLC39A8 (human solute carrier family 39 member 8) encodes a transmembrane protein that co-transports divalent heavy metal cations, such as Cd2+, with elusive physiological role. Recent genome-wide association studies have identified a non-synonymous single nucleotide polymorphism rs13107325 to be associated with hypertension. To investigate the functional impact of rs13107325 resulting in an amino acid substitution from Ala to Thr (A391T) in SLC39A8 on Cd2+ transport and the downstream signalling pathways. Design and method: Intracellular Cd2+ uptake was measured in HEK293 cells overexpressing SLC39A8 (Measure-iTTM Pb and Cd assay kit), and in human umbilical vascular endothelial cells (HUVECs) of different genotypes. Cd2+- and genotype-dependence of ERK1/2 and NF-kB pathways activation were investigated by immunoblotting and dual-luciferase reporter assay. Cytotoxicity was measured by the lactate dehydrogenase assay and MTS assay. Molecular dynamics simulations were performed to predict in silico the effect of A391T on the structure and dynamics of SLC39A8 by using Robetta, TMHMM and etc. Results: Overexpression of Ala variant in HEK293 resulted in higher Cd2+ uptake and higher cytotoxicity as compared with the Thr variant. This is associated with increased phosphorylation of ERK1 and NF-kB activation. Similar trends were observed in HUVECs with endogenous SLC39A8. Bioinformatics tools also suggested a conformational change of the &agr;-helical structural transition (residual 390–392) in the Thr mutant, which potentially attenuates the protein function. Conclusions: Increased Cd2+ uptake by SLC39A8 Ala variant (blood pressure raising allele) is associated with higher cell death in human kidney and endothelial cells. Therefore its altered function due to rs13107325 may indicate a potential therapeutic target in hypertension.

Chapter 23 – Genetic Markers in Prediction of Cardiovascular Disease

Cardiovascular disease is the number one contributor to mortality and morbidity world-wide and hypertension is responsible for 50% of its major risk factors with a significant heritable component. We review the current state of genomic discoveries of disease-causing variants and evaluate the progress made in improving therapeutic targeting, gene-based disease predictive tools, and stratified and personalised medicine approaches in relation to blood pressure and related phenotypes. Given that blood pressure regulation is both the causal factor as well as a consequence of early vascular aging and the genetic basis of blood pressure traits have been extensively studied, we focus on the progress toward understanding this common cause of stroke and coronary disease.

CHAPTER 41:Fructose Absorption and Enteric Metabolism

This chapter reviews the current understanding of how fructose is absorbed from the human diet and enters the circulation. Absorption of free fructose requires passage across the intestinal epithelium and is mediated by at least two facilitated hexose transporters, GLUT5 and GLUT2 which are expressed in both poles of epithelium. The activity of these transporters is regulated during the course of a meal by complex luminal sensing and enteric peptide control loops which match the absorptive capacity to the dietary load. When changes in the carbohydrate content of the diet are maintained over several days, this results in a resetting of the base level of transporter expression to provide a suitable safety margin. The fructose content and composition of Western diets has changed increasing significantly over the last 50 years. This raises questions as to the role this hexose may play in the current increase in reported incidence of intestinal complications and symptoms such as obesity, type II diabetes, and hypertension.