Asim K. Mandal (1), Megan P. Leask (3,4), Hyon K. Choi (5), Julia Horsfield, Tony R. Merriman (3,6), David B. Mount (1,2)
Affiliation(s):
1. Renal Divisions,1Brigham and Women’s Hospital and
2. VA Boston Healthcare System, Harvard Medical School, Boston, MA
3. Department of Physiology, University of Otago, Dunedin, New Zealand
4. Division of Rheumatology and Clinical Immunology, University of Alabama, Birmingham AL
5. Division of Rheumatology, Massachusetts General Hospital, Harvard Medical School, Boston MA
6. Department of Pathology, University of Otago, Dunedin, New Zealand
7. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
Early genome wide association studies (GWAS) identified TMEM171 (transmembrane protein 171) as a genetic determinant of SUA variation and the risk of gout. The causal genetic variant, rs17632159, is in fact intergenic, mapping between TMEM171 and the adjacent TMEM174 gene. We demonstrate here that the TMEM171 and TMEM174 proteins are novel physiological regulators of urate transport. Immunohistochemistry of human kidney indicates that TMEM17 protein is found in basolateral membrane and cytoplasm of proximal and distal tubule cells, whereas TMEM174 protein is selectively expressed at the apical membrane of proximal-tubule cells.
TMEM171 and TMEM174, expressed in transfected HEK 293T cells or Xenopus oocytes, did not show urate transport activity. However, both proteins efficiently inhibited the urate transport activities of URAT1, OAT10, OAT1, OAT3, ABCG2 and ABCC4 and inhibited the N-glycosylation of GLUT9 isoform proteins, without affecting the N-glycosylation of URAT1 or OAT10.
Both the TMEM171 and TMEM174 proteins co-immunoprecipitate with GLUT9 isoforms. However, unlike TMEM174, TMEM171 efficiently inhibits urate transport activities of GLUT9 isoforms and of GLUT9 N-glycosylation-deficient mutants, indicating that the inhibitory effect of TMEM171 is independent of its inhibitory effects on glycosylation.
The urate-increasing and gout risk allele (G) at rs17632159 is associated with lower expression of TMEM171 in esophagus mucosa in the Genotype-Tissue Expression Database (P=1.9x10-5). We also leveraged kidney expression quantitative trait locus (eQTL) data and found that rs17632159 was the lead associated SNP associated with TMEM171 expression in kidney tubules; again, the G-allele was also associated with lower renal tubular expression of TMEM171. Notably, rs17632159 is within the core motif of a predicted binding site for the CTCF transcription factor, and maps to a genomic location with enhancer histone marks in nine tissues, along with five bound transcription factor proteins (CTCF, RAD21, SMC3, ZNF263, NR2F1).
We tested the 818 bp segment of DNA containing rs17632159 for enhancer activity in human cell-based and zebrafish assays. We found significant enhancer activity of a construct containing the C-allele in HEK293 cells, consistent with this allele being associated with higher expression of TMEM171 in the kidney tubule.
In summary, we report that the TMEM171 and TMEM174 “digenic” locus is a novel regulator of urate homeostasis, through effects of both proteins on urate transporter activity. In particular, TMEM171 is a potent inhibitor of GLUT9-mediated urate transport.
The causal rs17632159 variant appears to selectively affect transcriptional regulation of the TMEM171 gene, with the urate-decreasing allele increasing expression of TMEM171 and thus amplifying the inhibitory effect of TMEM171 on GLUT9 function and proximal tubular urate reabsorption.