Function and localization of urate transporter 1 in mouse kidney.

Abstract

Mouse renal-specific transporter (RST) cDNA, the amino acid sequence of which has 74% identity with that of human urate transporter 1 (hURAT1), is potentially the mouse homologue of hURAT1, the gene responsible for hereditary renal hypouricemia. The aim of this study is to determine the location and characteristics of RST molecule in mouse kidney and investigate urate transport by RST using the Xenopus oocyte expression system. RST transported (14)C-urate in a Michaelis-Menten manner. The K(m) and the V(max) values of RST-dependent urate transport were 1213 +/- 222 micro M and 268.8 +/- 38.0 pmol/oocyte per hr, respectively (n = 3). RST-dependent urate transport was cis-inhibited significantly by 1 mM probenecid (68.7 +/- 9.4%), 50 micro M benzbromarone (67.9 +/- 6.4%), and 10 mM lactate (50.9 +/- 9.5%). However, 1 mM p-aminohippurate (PAH), 1 mM xanthine, and 1 mM oxonate did not inhibit RST-dependent urate transport. Substitution of Cl anion with gluconate in the external solution enhanced RST-dependent urate transport. Pre-injected pyrazinoic acid (PZA) or L-lactate trans-stimulated RST-dependent urate transport. Using immunohistochemistry for mouse kidney, the brush border or intracellular membrane of proximal tubules was stained by an affinity-purified antibody that recognized mouse URAT1 (mURAT1) expressed on Xenopus oocyte. Using Western blotting, anti-mURAT1 antibody detected 70-kD and 62-kD protein bands. The 70-kD protein was N-glycosylated and was identified as a Triton X-100 insoluble brush border membrane protein. RST mRNA and protein levels were higher in male kidneys than female. RST transported urate similar to hURAT1 and, therefore, appears to be mURAT1-the mouse homologue of hURAT1.