Identification of Transporters Involved in Drug-drug Interactions During Gout Treatment in Primary Rat Hepatocytes

Abstract

Gout is one of the most common form of inflammatory arthritis, with hyperuricemia as the major risk factor. Chronic hyperuricemia, or high level of serum uric acid (SUA), can lead to the formation of monosodium urate crystals (MSU) in the joints, which can result in acute flares in gout. Allopurinol is the gold standard therapy for gout, with its active metabolite, oxypurinol, acts to inhibit the enzyme responsible for uric acid synthesis, xanthine oxidase (XO), thus lowering SUA level. Moreover, ~ 70% of US adults with gout also has hypertension, which is often treated with diuretics such as furosemide. However, concomitant treatment with furosemide compromises the therapeutic effects of allopurinol. The molecular mechanisms underlying this adverse drug-drug interaction are unknown. Based on current knowledge of the transport of furosemide and allopurinol/oxypurinol by transporters in the kidney, and the fact that similar transporter setups exists in the liver, as well as evidence from clinical studies, we hypothesise that transporters known to translocate these drugs in the kidney are responsible for the drug-drug interactions in the liver, where allopurinol/oxypurinol act to lower SUA. Hence, the aim of this project was to mimic the in vivo situation in gout patients with our cell model by treating cultured primary rat hepatocytes with gout-associated drugs. The functional outputs were assessed by measuring extra- (EUA) and intracellular uric acid (IUA) level.

First, we were able to successfully establish a protocol to extract primary hepatocytes via in situ perfusion method. These extracted cells had polygonal morphology, characteristic of primary hepatocytes described in the literature. We characterised the gene expression profile of urate transporters and its converting enzymes in these hepatocytes and in the rat liver tissue. In both the extracted hepatocytes (n=3) and the tissue (n=6), qPCR analysis confirmed expression of the following genes: AOX1, XO, Oat2, Oat3, Glut9, Mrp4, Npt1, Npt4, and Abcg2. Furthermore, immunoblotting was carried out to confirm protein expression of our main proteins of interest: XO, Oat2, Glut9, Mrp4, and Abcg2. However, a temporal profile of the gene expression of the hepatocytes found that, after 48h there was a downregulation of most of the genes, except for Npt4 and Mrp4, which seemed to have not changed, and Abcg2 seemed to be upregulated (n = 1). From functional studies, we treated the cells with combinations of 250 μM allopurinol, 250 μM oxypurinol, 1 mM furosemide, and 1 mM probenecid. 24h after treatment, the cells and media were harvested for analysis. Our results showed that there was a significant decrease in EUA in cells treated with probenecid, which was in agreement with our model. However, due to low sample numbers, we were not able to draw any conclusion from these functional results. Additionally, a knockdown study was performed to evaluate the contribution of Oat2 and Glut9 to the transport of allopurinol and oxypurinol. Results from one set of experiment suggest that Oat2 might play a bigger role in transport of these drugs than Glut9. Further experiments are required to confirm transport of allopurinol/oxypurinol by these two transporters.