Background: Soluble uric acid stimulates vascular smooth muscle cell (VSMC) proliferation by activating mitogen-activated protein kinases, and stimulating COX-2 and PDGF synthesis. The mechanism by which uric acid enters the VSMC is not known. We hypothesized that uric acid enters via transporters similar to that observed in the kidney. Methods: We studied the uptake of uric acid into rat VSMC under polarized and depolarized conditions and in the presence of organic anion transport (OAT) inhibitors (probenecid and benzbromarone) or p-aminohippurate (PAH). We also examined the ability of probenecid to inhibit uric acid-induced VSMC proliferation and monocyte chemoattractant protein-1 (MCP-1) synthesis. Results:¹⁴C-Urate uptake was shown in VSMC and was enhanced under depolarized conditions. ¹⁴C-Uric acid uptake was inhibited by probenecid and benzbromarone, as well as by unlabelled urate and PAH. Probenecid blocked VSMC proliferation and MCP-1 expression in response to uric acid. VSMC did not express rOAT1-3, rOAT-5 or URAT-1 mRNA by PCR, but did express the voltage-sensitive transporter (UAT) by both PCR and RNase protection assay. Conclusions: Urate enters VSMC by both voltage-sensitive and OAT pathways, and the uptake, cell proliferation and MCP-1 expression can be blocked by OAT inhibitors. The specific transporter(s) responsible for the urate uptake remains to be determined.

Maintenance of urate homeostasis requires urate efflux from urate-producing cells with subsequent renal and gastrointestinal excretion. The molecular basis for urate transport, however, has not been identified. A novel full-length cDNA encoding a 322-amino acid protein, designated UAT (urate transporter), has been cloned from a rat renal cDNA library by antibody screening. UAT mRNA transcripts that approximate 1.55 kilobases are present, but differentially expressed in various rat tissues. Recombinant UAT protein that was expressed from the cloned cDNA in Escherichia coli and purified via immobilized metal affinity chromatography has been functionally reconstituted as a highly selective urate transporter/channel in planar lipid bilayers. The IgG fraction of the polyclonal antibody that was used to select the UAT clone from the cDNA library, but not nonimmune IgG, blocked urate channel activity. Based on the wide tissue distribution of the mRNA for UAT we propose that UAT provides the molecular basis for urate flux across cell membranes, allowing urate that is formed during purine metabolism to efflux from cells and serving as an electrogenic transporter that plays an important role in renal and gastrointestinal urate excretion.

Recombinant protein, designated UAT, prepared from a cloned rat renal cDNA library functions as a selective voltage-sensitive urate transporter/channel when fused with lipid bilayers. Since we previously suggested that UAT may represent the mammalian electrogenic urate transporter, UAT has been functionally characterized in the presence and absence of potential channel blockers, several of which are known to block mammalian electrogenic urate transport. Two substrates, oxonate (a competitive uricase inhibitor) and pyrazinoate, that inhibit renal electrogenic urate transport also block UAT activity. Of note, oxonate selectively blocks from the cytoplasmic side of the channel while pyrazinoate only blocks from the channel's extracellular face. Like oxonate, anti-uricase (an electrogenic transport inhibitor) also selectively blocks channel activity from the cytoplasmic side. Adenosine blocks from the extracellular side exclusively while xanthine blocks from both sides. These effects are consistent with newly identified regions of homology to uricase and the adenosine A1/A3 receptor in UAT and localize these homologous regions to the cytoplasmic and extracellular faces of UAT, respectively. Additionally, computer analyses identified four putative alpha-helical transmembrane domains, two beta sheets, and blocks of homology to the E and B loops of aquaporin-1 within UAT. The experimental observations substantiate our proposal that UAT is the molecular representation of the renal electrogenic urate transporter and, in conjunction with computer algorithms, suggest a possible molecular structure for this unique channel.

The molecular definition and localization of four urate transport proteins provides a basis for developing a molecular model of the bi-directional transport of urate in renal proximal tubules. It seems likely that the urate-anion exchanger is responsible for luminal reabsorption while the urate transporter/channel permits secretion of urate from the cell into the lumen. Since organic anion transporters 1 and 3 reside in the basolateral membrane, one or both may be relevant in the reabsorptive flux of urate into the peritubular capillary as well as in the cellular uptake of urate from the peritubular space, the first step in the process of urate secretion. Knowledge of the molecular basis of urate transport should provide greater insights into states of altered transport as well as assist in development of drugs to modify urate flux.