Expression of SLC2A9 Isoforms in the Kidney and Their Localization in Polarized Epithelial Cells

Abstract

BackgroundMany genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro.Methodology/Principal FindingsIsoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type.Conclusions/SignificanceSLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking.

Structure of, and functional insight into the GLUT family of membrane transporters

“…This observation is supported by reports of patients, primarily from Japan, who have mutations in the hSLC2A9 gene. 28 Unlike the majority of mammals, humans maintain high plasma levels of urate, as a result of their modified metabolic pathway that is missing the enzyme uricase. Consequently, this metabolite is retained in the body as opposed to being broken down to allantoin and lost in the urine.…”

“…This GLUT can transport both glucose and fructose when expressed in Xenopus oocytes with high affinity, but with very low transport capacity, and it is not sensitive to cytochalasin B. 26,28 It is likely that its correct physiological substrate has yet to be identified (see GLUT9 below).…”

“…28 It shares 38% and 44% identity with GLUT1 and GLUT5, respectively. Human GLUT9 was initially shown to be a high affinity/low capacity glucose and fructose transporter, but more recently as a consequence of a series of genome-wide association studies (GWAS) was identified as a high capacity urate transporter.…”

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

“…Many of these glucose transporter proteins are expressed specifically in different human tissues. For instance, the human glucose transporter 9 (hSLC2A9) is found primarily in the human kidney and liver (5,6). Genome-wide association studies suggest that nucleotide polymorphisms within hSLC2A9 are associated with uric acid (urate) handling and urate-related diseases in humans, such as gout, diabetes, and hypertension (7)(8)(9)(10)(11)(12).…”

Uric acid transporter inhibitors for gout

“…A short isoform of GLUT9 (GLUT9b or -S) differs at the amino terminus from the longer isoform due to alternative splicing. An antibody raised against the unique 21 amino terminal amino acids of GLUT9b detected this variant on membranes of epithelial cells in the collecting duct [23], suggesting the possibility that this distal segment of the nephron handles urate. Interestingly, this antibody did not detect GLUT9b on the proximal tubule.…”