Zebrafish (Danio rerio) Oat1 and Oat3 transporters and their interaction with physiological compounds.

Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Km = 11.4 µM) and 6-carboxyfluorescein as Oat3 model substrate (Km = 5.8 µM). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.

Urolithiasis and osteoporosis: clinical relevance and therapeutic implications.

Several clinical and epidemiological studies revealed increased bone turnover and lower bone mass in patients with urolithiasis. Bone mass loss is particularly evident in idiopathic calcium stone formers. However, pathogenetic mechanisms and factors implicated in bone loss in these patients are still unknown. Dietary calcium restriction, increased intake of salt and animal proteins, vitamin D receptor polymorphisms are likely risk factors, while role of inflammatory cytokines, osteopontin and prostaglandin mediated bone resorption is yet to be determined. Regarding treatment and prevention, it has been proven that calcium supplements and high calcium diet with the addition of potassium alkali have an important role in prevention and treatment of both, urolithiasis and osteoporosis. Thiazide diuretics reduce hypercalciuria in renal tubules, and in addition promote osteoblast differentiation. Finally, bisphosphonates, a commonly used drugs in treatment of osteoporosis, show the potential to inhibit calcium stone formation, whereas a possible protective effect of antioxidants in bone loss and renal injurie needs to be investigated further.